Original Article

Strategic self-limiting production

of infectious HIV particles
by CRISPR in permissive cells
Hong Liu,1,7 Chen Chen,1,7 Shuren Liao,1 Danielle K. Sohaii,2 Conrad R.Y. Cruz,2 Tricia H. Burdo,1
Thomas J. Cradick,3 Anand Mehta,4 Carlos Barrero,5 Magda Florez,5 Jennifer Gordon,3 Stephane Grauzam,4
James Dressman,4 Shohreh Amini,6 Catherine M. Bollard,2 Rafal Kaminski,1 and Kamel Khalili1
1Center for Neurovirology and Gene Editing, Department of Microbiology, Immunology, and Inflammation, Lewis Katz School of Medicine at Temple University, 3500 N.
Broad Street, 7th Floor, Philadelphia, PA 19140, USA; 2Center for Cancer and Immunology Research, Children’s National Health System, The George Washington
University, 7144 13th Place NW, Washington, DC 20012, USA; 3Excision Biotherapeutics, Inc., 499 Jackson Street, San Francisco, CA 94111, USA; 4Department of Cell
and Molecular Pharmacology, Medical University of South Carolina, Basic Science Building, Room 310, 173 Ashley Avenue, Charleston, SC 29425, USA; 5Department
of Pharmaceutical Sciences, School of Pharmacy, Temple University, 3307 N. Broad Street, Philadelphia, PA 19140, USA; 6Department of Biology, College of Science
and Technology, Temple University, 1900 North 12th Street, Philadelphia, PA 19122, USA

Post-translational glycosylation of the HIV-1 envelope protein
involving precursor glycan trimming by mannosyl oligosaccha-
ride glucosidase (MOGS) is critically important for morpho-
genesis of virions and viral entry. Strategic editing of the
MOGS gene in T lymphocytes and myeloid origin cells
harboring latent proviral DNA results in the production of
non-infectious particles upon treatment of cells with latency
reversal agents. Controlled activation of CRISPR-MOGS by
rebound HIV-1 mitigates production of infectious particles
that exhibit poor ability of the virus to penetrate uninfected
cells. Moreover, exclusive activation of CRISPR in cells infected
with HIV-1 alleviates concern for broad off-target impact of
MOGS gene ablation in uninfected cells. Combination
CRISPR treatment of peripheral blood lymphocytes prepared
from blood of people with HIV-1 (PWH) tailored for editing
the MOGS gene (CRISPR-MOGS) and proviral HIV-1 DNA
(CRISPR-HIV) revealed a cooperative impact of CRISPR treat-
ment in inhibiting the production of infectious HIV-1 parti-
cles. Our design for genetic inactivation of MOGS by
CRISPR exhibits no detectable off-target effects on host cells
or any deleterious impact on cell survival and proliferation.
Our findings offer the development of a new combined gene ed-
iting-based cure strategy for the diminution of HIV-1 spread
after cessation of antiretroviral therapy (ART) and its elimina-
tion.
INTRODUCTION
Entry of human immunodeficiency virus type-1 (HIV-1) to host cells,
including T lymphocytes and myeloid lineage cells, is a multistep pro-
cess that begins with the interaction of the glycosylated viral envelope
protein, gp120 (Env), with host cell receptors, CD4, and co-receptors,
including CCR5 and CXCR4,1,2 followed by fusion of viral and host
cell membranes,3 internalization of the virion, and release of its
genomic RNA in the cytoplasm.4 Both gp120 and gp41 subunits of
HIV-1 Env are N-linked glycosylated, with 28 of 32 potential
N-linked glycosylation sites (PNGs) located in gp120.5–8 The extensive
glycosylation of Env shields the viral envelope protein from recogni-
tion by host neutralizing antibodies.9–12 N-linked glycosylation is a
multistep post-translational process in which a high mannose-type
sugar chain (Glc3Man9GlcNAc2) is engaged with an asparagine resi-
due in the context of the conserved motif Asn-X-Ser/Thr, where X
can be any amino acid except proline.13 This intracellular chemical
modification is followed by trimming of glucose and mannose residues
and adding terminal sugars to constitute a complex-type glycan as the
glycoprotein structurally matures through the endoplasmic reticulum
(ER) and Golgi along cellular secretory pathways.13,14 MOGS (manno-
syl oligosaccharide glucosidase) is the ER-resident enzyme of the
N-linked glycosylation pathway responsible for the removal of the first
glucose residue from the precursor glycan an indispensable step
required for further trimming and re-decoration of glycan chains of
nascent glycoprotein in ER-lumen before turning to Golgi apparatus
in the cytosol (Figure S1A). These observations prompted us to postu-
late that ablation of the MOGS gene by CRISPR gene editing either
alone or in combination with CRISPR-mediated editing of HIV-1
Received 15 December 2022; accepted 28 April 2023;
https://doi.org/10.1016/j.omtn.2023.04.027.
7
These authors contributed equally
Correspondence: Rafal Kaminski, Center for Neurovirology and Gene Editing,
Department of Microbiology, Immunology, and Inflammation, Lewis Katz School
of Medicine at Temple University, 3500 N. Broad Street, 7th Floor, Philadelphia, PA
19140, USA.
E-mail: rafalkim@temple.edu
Correspondence: Kamel Khalili, Center for Neurovirology and Gene Editing,
Department of Microbiology, Immunology, and Inflammation, Lewis Katz School
of Medicine at Temple University, 3500 N. Broad Street, 7th Floor, Philadelphia, PA
19140, USA.
E-mail: kamel.khalili@temple.edu

1010 Molecular Therapy: Nucleic Acids Vol. 32 June 2023 ª 2023

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.moleculartherapy.org

(legend on next page)

Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1011

 Molecular Therapy: Nucleic Acids

proviral DNA mitigates the production of fully infectious particles through the cell cycle, and apoptosis were noted in MOGS
T
from the primary cells (Figure S1B). lymphoid and myeloid cells (Figures S6A–S6D; also see Table S3).

RESULTS Impact of CRISPR-MOGS treatments on secondary infection of

Inactivation of MOGS by CRISPR gene editing
The initial bioinformatic assessment of the human MOGS gene
sequence led to the identification of two potential binding sites for
CRISPR gRNAs, designated A and B (Figures S2A and S2B) with
high on-target and minimum, if any, nominated off-target activity
in the host genome (Table S1). Protospacer regions of these gRNAs
were sequentially cloned into “all-in-one” AAV-delivery SaCas9
pX601 vector, hereafter referred to as CRISPR-MOGS (Figure S2C),
and expression of each gRNA and SaCas9, and the excision of the
MOGS gene was verified (Figures S2D and S2E).
To assess the importance of MOGS in the genesis of infectious virions,
in the first proof-of-principle experiments, we used the TZM-bl cells,
a HeLa-CD4/CCR5/CXCR4 laboratory cell model that supports viral
replication.15,16 CRISPR-MOGS-treated TZM-bl cells were clonally
expanded and single-cell clones with a complete knockout of the
MOGS gene were identified. The introduction of CRISPR-MOGS
plasmid to TZM-bl cells resulted in the precise removal of the as-
signed DNA fragment spanning between the A and B target sites in
the MOGS gene (Figure 1A) and was verified using Sanger DNA
sequencing (Figures 1B and 1C). As expected, results from the
RNA and protein detection assays showed a lack of expression of
the MOGS gene (Figures 1D and 1E). Investigation of HIV-1 replica-
tion by detecting the reporter GFP marker protein in the HIV-1NL4-3-
GFP-P2A-nef-infected cells (Figure 1F), and the Gag p24 viral protein
levels in the culture media of the primary infection (Figure 1G) indi-
cated that initially (days 1 and 2) the elimination of MOGS in TZM-bl
had virtually no impact on the primary infection. At day 3 post-infec-
tion, virus levels in MOGS+ cells increased while its levels in MOGS

cells remain unchanged. Under similar experimental conditions, it
was noted that the viral progenies from the MOGS
cells are inca-
pable of replicating in the secondary infection, suggesting that aber-
ration in trimming of heavily glycosylated viral gp120 in the absence
of MOGS results in the production of the larger size of viral glycopro-
tein (Figure 1H), which is associated with the decline in the viral
infectivity (Figure 1I). Importantly, as shown in Figures S3 and S4
and Table S1, the introduction of CRISPR-MOGS to TZM-bl cells
had no impact on the top five bioinformatically nominated off-target
sites in the human genome. Also, no adverse effects on cell viability
and growth rate (Figures S5A–S5D; also see Table S3), the progression
HIV-1 in T lymphocytes and latently infected myeloid cells
As CD4+ T cells are the major targets for HIV-1, we verified the
effects of MOGS knockout on HIV-1 infection of Jurkat
T-lymphoid cell line and primary CD4+ T cells. Similarly, as
observed in MOGS
TZM-bl cells, HIV-1 infection of MOGS
Ju-
rkat cells resulted in a shift in the size of the viral envelope glyco-
protein (Figure 2A; also see Figure 1H), with no impact on the level
of the viral p55 capsid protein and no drastic effect on the primary
viral infection (Figures 2B and 2C). However, viral progeny pro-
duced in these cells was completely non-infectious (Figure 2D).
This result once again points to the importance of MOGS in the
genesis of infectious viral particles.
We then used primary human CD4+ T cells isolated from the pe-
ripheral blood of healthy donors. Cells were electroporated with
CRISPR ribonucleoprotein complexes composed of Cas9 and four
different gRNAs (MOGS#1–4) targeting the coding sequence of
the MOGS gene and then infected with HIV-1NL4-3-Bal-GFP. The
genomic DNA and protein analyses revealed that treatment of cells
with gRNAs MOGS#1 and #4, which showed the highest on-target
indel knockout scores (Figure S7), led to the most profound
decrease in MOGS protein level (Figure 2H) compared with control
or gRNA MOGS#2 and #3 treated cells. Importantly, similarly, as in
the cell line experiments, we did not observe differences in primary
infection levels in RNP-CRISPR-MOGS-treated CD4+ T cells
compared with control cells (Figures 2F and 2G). On the other
hand, the virus present in the supernatants from these cells was
significantly less infective in the second round of infection using
target HutR5 cells (Figure 2H). Furthermore, the secondary infec-
tion levels correlated with the levels of MOGS expression observed
in primary CD4+ T cells, i.e., more MOGS protein expression (Fig-
ure 2E, #2 and #3), the less suppression of viral presence in the sec-
ondary infection (Figure 2H, #2 and #3).
Recently, myeloid cells have received more attention because of their
potential role in the pathogenesis of HIV infection in hosting/main-
taining HIV-1 in the latent stage.17,18 Here, we found that treatment
of the latently infected myeloid cells (U1) with CRISPR-MOGS fol-
lowed by reactivation of the latent HIV-1 using PMA plus the
HDAC inhibitor Vorinostat (SAHA) showed a clear shift in the size

Figure 1. Editing of the MOGS gene by CRISPR affects the infectivity of the viral progeny derived from MOGS– cells
(A) PCR genotyping results of CRISPR-Cas9-mediated excision of an exon 1 and 2 region of the MOGS gene in clone 2 of TZM-bl cells. WT TZM-bl cells were used as a
control. The arrows point to intact full-length 1,620-bp-long MOGS promoter-intron 3 amplicon (top arrow) and CRISPR-cleaved/end-joined 433 bp truncated amplicon
(bottom arrow), carrying 1,187 bp deletion of the exon 1 and 2 region of the MOGS gene. (B) Sanger sequence tracing of truncated CRISPR-cleaved/end-joined amplicons
detected for MOGS
TZM-bl clone 2. (C) Sequence alignment of the same sequences. gRNAs target sites are highlighted in green and PAM sequences in red. (D) qRT-PCR
analysis of MOGS mRNA levels in WT and MOGS
clone 2 TZM-bl cells. b-Actin mRNA was used as a reference gene. (E) Immunoblot analysis of MOGS protein expression
in the same cells. a-tubulin was used as a loading control. (F) GFP-flow cytometry analysis of HIV-1NL4-3-GFP infection in WT and MOGS
clone 2 TZM-bl cells. (G) HIV-1 gag
p24 ELISA results for supernatants collected from the same cells. (H) Immunoblot analysis of HIV-1 gp160 in infected WT and MOGS
clone 2 TZM-bl cells. PNGaseF
digestion was used to remove glycans from glycoproteins in lysates. HIV-1 p55 and a-tubulin were used as infection and loading controls, respectively. (I) GFP-flow cytometry
analysis of HutR5 T-lymphoid cells infected with supernatants derived from HIV-1NL4-3-GFP-infected cells from (G). Student’s t test (two-tailed) was used in (I). ****p < 0.0001.

1012 Molecular Therapy: Nucleic Acids Vol. 32 June 2023

www.moleculartherapy.org

Figure 2. The MOGS gene knockout in T and monocytic cells results in the production of infection-defective progeny virions
(A) Immunoblot analysis of HIV-1 gp160 and MOGS in HIV
1NL4-3-GFP-infected WT and MOGS
clone 5 and 13 Jurkat cells. HIV-1 p55 and a-tubulin were used as infection
and loading controls. (B) GFP-flow cytometry analysis of HIV-1NL4-3-GFP infection in WT and MOGS
clone 5 and 13 Jurkat cells. (C) HIV-1 gag p24 ELISA results for su-
pernatants collected from the same cells. (D) GFP-flow cytometry analysis of HutR5 T-lymphoid cells infected with supernatants derived from HIV-1NL4-3-GFP-infected WT and
MOGS
clone 5 and 13 Jurkat cells. (E) Immunoblot analysis of MOGS in primary human CD4+ T cells electroporated with RNP-CRISPR-Cas9/MOGSgRNA #1, #2, #3, or
#4 complexes. WT cells were used as a control. a-tubulin was used as a loading control. (F) ddPCR quantification of viral DNA in HIV-1NL4-3-GFP-infected cells from (E). (G)
HIV-1 gag p24 ELISA results for supernatants collected from HIV-1NL4-3-GFP infected primary human CD4+ T cells. (H) ddPCR quantification of viral DNA in HutR5 T-lymphoid
cells infected with supernatants derived from HIV-1NL4-3-GFP infected WT and RNP-CRISPR-Cas9/MOGSgRNA #1, #2, #3, or #4 treated primary human CD4+ T cells. (I)
Immunoblot analysis of HIV-1 gp160 and MOGS in uninduced or PMA/SAHA-treated U1 WT and MOGS
cells. HIV-1 p55 and a-tubulin were used as infection and loading
controls. (J) HIV-1 gag p24 ELISA results for supernatants collected from cells in (I). (K) ddPCR quantification of viral DNA in HIV-1 permissive HutR5 T-lymphoid cells infected
with supernatants derived from cells in (I). One-way ANOVA and Dunnett’s multiple comparisons test was used in (H). ****p < 0.0001.

Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1013

 Molecular Therapy: Nucleic Acids

Figure 3. Viral particles released from MOGS– cells fail to enter uninfected T cells
(A) Electron microscopy images of HutR5 cells incubated for 1 h with equal titers of virus-containing supernatants derived from WT (A1–A4) or MOGS
(B1–B4) J1.1 cells.
Original images with scale bars and acquisition details are shown in Figure S8. (B) Images of TZM-bl cells loaded with the CCF2-AM BlaM substrate after inoculation with HIV
virus bearing the BlaM-Vpr chimera produced from HEK293T WT cells or MOGS
cells (clone 4). Cells were identified by CCF2-AM signal (488/555nm, green), and viral entry
was detected by intracellular processed-CCF2 signal (400/430nm, blue). Scale bars 50 mm. (C) Bar graph showing the percentage of viral entry (cleaved-CCF2-positive) with
respect to the total number of cells per well (uncleaved-CCF2-AM-positive). ****p < 0.0001. Data were analyzed using the two-tailed unpaired t test, using Prism software.

of the viral envelope glycoprotein produced in CRISPR-MOGS-
treated U1 cells, indicative of misconfiguration of glycans of the viral
envelope protein emerged from the MOGS
myeloid cells (Figure 2I).
As before, treatment did not affect the level of viral production in the
parental cells, yet the reactivated viral particles were incapable of
spreading and completing secondary infection in naive, permissive
HutR5 cells (Figures 2J and 2K).
On the basis of the fact that the persistent HIV-1 reservoir responsible
for the virus reemergence upon antiretroviral therapy (ART) inter-
ruption is predominantly composed of latently infected resting mem-
ory CD4+ T cells,19 in the next set of experiments, we used two well-
characterized HIV-1 latency T cell line models: ACH-2 and J1.1
cells.20–22 Same as before, cells were treated with CRISPR-MOGS,
and single-cell MOGS
clones were identified (Figure 4A). In

1014 Molecular Therapy: Nucleic Acids Vol. 32 June 2023

www.moleculartherapy.org

Figure 4. Upon latency reversal, virions released from MOGS– latently infected T cells show residual infectivity and contain hyper-glycosylated
(unprocessed) glycans
(A) Immunoblot analysis of HIV-1 gp160 and MOGS expression in WT and MOGS
ACH-2 and J1.1 latently infected T cell lines upon PMA/SAHA (ACH-2) or TNF-a (J1.1)
treatment. a-Tubulin was used as the loading control. (B) ddPCR analysis of genomic DNA extracted from HutR5 cells treated with supernatants derived from cells from (A).
(C) Immunoblot analysis of HIV-1 gp160 in the untreated or treated with Endo H or PNGaseF protein lysates prepared from WT and MOGS
ACH-2 and J1.1 cells (same as in
A). HIV-1 p55 was used to show viral protein expression levels. (D) Viral particles from supernatants of PMA/SAHA or TNF-a-treated wild-type cells (ACH- 2 and J1.1) or
MOGS
(ACH-2 C17 and C20, J1.1, C8, and C12) cells were purified and analyzed using MALDI-glycan imaging.24 N-linked glycan consisting of large structures with m/z
values corresponding to Hexose11HexNAc2 (m/z = 2,279.7400) and Hexose12HexNAc2 (m/z = 2,391.7929) were observed only in the MOGS
cells.

follow-up studies, supernatants from MOGS+ and MOGS
cells
were collected and used to infect lymphocytes. In the first study,
HutR5 T cells were incubated for 1 h with equal titers of virus ob-
tained from the supernatants collected from MOGS+ and MOGS

J1.1 cells (300 ng Gag p24/107 cells) and, after fixing, analyzed using
electron microscopy for the presence of cell membrane-associated vi-
rions. Viral particles obtained from MOGS+ cells are scattered
around the cell membrane (Figure 3A, sub-panels A1 and A2; also
see Figures S8A and S8B), and that a large number of viral particles
are detected in the cytoplasm, suggesting successful viral entry (Fig-
ure 3A, sub-panels A3 and A4; also see Figures S8C and S8D). In
several instances, the attachment of the virus to the target cells was
clearly detected (see Figure S8). Interestingly, we observed an unusual
assembly of viruses obtained from MOGS
cells, as they appeared to
be lined up outside of the uninfected cells (Figure 3A, sub-panels B1
and B2; also see Figures S8E and S8F). Furthermore, many of the cells
exhibited the limited presence of the virus inside of the cells (Fig-
ure 3A, sub-panels B3 and B4; also see Figures S8G and S8H). These
results are in accord with the observations in Figure 2, pointing to the
weak infectivity of HIV particles produced in the MOGS
cells. To
further investigate the viral entry step, we performed a sensitive
and specific enzyme-based assay allowing quantitative measurement
of virion entry by fusion, Vpr-b-lactamase assay.23 In this assay b-lac-
tamase-Vpr chimeric proteins (BlaM-Vpr) are incorporated during
packaging into HIV-1 virions and subsequently delivered into the
cytoplasm of the target cells upon successful fusion-dependent viral
entry. Prior to infection, target cells are loaded with a fluorescent sub-
strate of b-lactamase (BlaM), the CCF2 dye which is cleaved by BlaM,

Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1015

 Molecular Therapy: Nucleic Acids

(legend on next page)

1016 Molecular Therapy: Nucleic Acids Vol. 32 June 2023

www.moleculartherapy.org

changing the fluorescence emission spectrum from green (520 nm) to
blue (447 nm) and thereby allowing viral entry to be detected by fluo-
rescence microscopy. Results from this assay (Figures 3B and 3C)
showed significantly lower levels of viral entry by HIV-1 virions
collected from the MOGS
cells compared with those that are pre-
pared from the MOGS+ cells under comparable conditions
(Figures 3B and 3C; also see Figure S9). Altogether, the results from
electron microscopy and viral entry assays point to the inability of
the virus derived from the MOGS
cells to penetrate uninfected cells
which result in a block of HIV-1 infection cycle and virus spread.
Next, we investigated the state of HIV-1 Env glycosylation and the
importance of MOGS in the infectivity of emerging rebound viruses
using MOGS
J1.1 and ACH-2 cells. The lack of MOGS expression
and the subsequent shift in the size of viral envelope protein upon vi-
rus reactivation with latency reversal drugs was confirmed using west-
ern blot (Figures 4A, 4C, and S10). Results from secondary infection
verified a drastic decline in the infectivity of the rebound virus that
was reactivated in cells with no MOGS expression (Figure 4B).
Furthermore, the differences in molecular weight depended on
N-glycosylation pattern, as revealed after N-glycan removal by pre-
treatment of protein lysates with Endo H that removes glycan and
PNGase F that removes N-linked oligosaccharides from glycoprotein
(Figure 4C; also see Figure S10).
Subsequently, the glycan analysis of the viruses purified from the
MOGS
cells was performed. Purified virus (see materials and
methods) was spotted onto nitrocellulose as described previously.25
Subsequently, glycan attached to the virus was analyzed by matrix-as-
sisted laser desorption ionization (MALDI)-glycan imaging.
Although complex glycans were detected in all samples (data not
shown), large glycans containing R10 hexoses with only two
HexNAcs were observed only on viruses produced in MOGS
cells
(Figure 4D). On the basis of the observed m/z values (2,279.7400
and 2,391.7929), these glycans are presumed to be a Glc3Man8 and
a Glc3Man9 glycan, which is the result of the inhibition of glucosidase
1 and 2. Additionally, the impact of MOGS deletion on the total
glycan profile on cell level was determined by MALDI-glycan imag-
ing. Briefly, MOGS+ (J), MOGS
(JM) J1.1 cells and MOGS+ (U),
MOGS
(UM) U1 cells were precipitated onto amine reactive slides
as described previously,26 and the glycosylation was determined
following application of PNGase F PRIME and MALDI imaging.27
We also observed large glycans containing R10–12 hexoses with
only two HexNAcs remaining in MOGS
cells (Figure S11). On
the basis of the canonical rules of N-linked glycosylation, the struc-
ture in Figure S11A, with a m/z of 2,391.7929, is a glycan composed
of 12 hexoses and 2 HexNAc, and would presumably have a structure
of a Glc3Man9GlcNAc2 glycan. Similarly, the glycans found in
Figures S11B and S11C correspond to Glc3Man8GlcNAc2 and
Glc3Man7GlcNAc2 glycan. The relative contribution of different
types of glycans to cell surface glycome of wild-type (WT) and
MOGS
cells is summarized in Figures S11D–S11G. It is noted
that glycan processing still occurred in these cells (Figures S11F and
S11G) and resulted in the formation of complex glycan, presumably,
through the use of the Golgi endo-a-mannosidase pathway.28
Strategic activation of CRISPR-MOGS by HIV-1 results in the
diminution of infectious particles
To restrict the editing of the MOGS gene to the HIV-1 infected cells,
we employed the strategy that triggers activation of CRISPR-MOGS
and the expression of its Cas9 endonuclease by the HIV transcription
trans-activator Tat.29 In the first set of experiments, we demonstrated
that a minimal sequence of the HIV-1 long terminal repeat (LTR)
promoter spanning
80 to +60 of the LTR remains responsive to
Tat protein in HEK293T cells (Figure S12A). Next, we showed that
Tat-mediated induction of CRISPR-MOGS results in the appearance
of larger size envelope proteins (Figure S12B) because of aberrant
glycan trimming and production of virions that are less infectious
during secondary infection (Figure S12D). Importantly, HIV-1 infec-
tion-derived Tat was sufficient to induce these changes (Figure S12B,
middle line). However, the level of the aberrantly glycosylated viral
envelope was higher upon overexpression of Tat (Figure S12B, third
line). Again, no impact on the primary infection (Figure S12C) was
observed. In subsequent studies, we found that similar to HEK293T
cells, Tat-mediated expression of SaCas9 in TZM-bl cells (Figure 5A),
by potentiating CRISPR-MOGS, results in the diminution of MOGS
protein and the appearance of the shifted (larger size) band, indicative
of aberrant trimming process of the viral envelop protein glycans in
the absence of MOGS in the infected cells (Figure 5D). This cascade
of events leads to the production of viral progeny with a significantly

Figure 5. Treatment of primary PWH-derived CD4+ T cells with Tat-inducible CRISPR-MOGS vector leads to self-limiting viral rebound and blocks the
spread of HIV infection
(A) Immunoblot analysis of SaCas9-HA expression in TZM-bl cells transfected with pLTR
80/+60-SaCas9-MOGS, pLTR
120/+60-SaCas9-MOGS, or pLTR
454/+60-
SaCas9-MOGS constructs together with empty pCMV (Tat
) or pCMV-Tat (Tat+) plasmids. a-Tubulin was used as an internal control. (B) GFP-flow cytometry analysis of
HIVNL4-3-GFP-infected cells. (C) GFP-flow cytometry analysis of HutR5 T-lymphoid cells infected with supernatants derived from infected cells shown in (B). (D) Immunoblot
analysis of gp160 in HIV-1NL4-3-GFP-infected TZM
bl cells untransfected (control) or transfected with pLTR
80/+60 SaCas9-MOGS in the absence or presence of Tat-
expressing plasmid. HIV-1 p55 and a-tubulin were used as infection and loading controls. (E) HIV-1 Gag p24 ELISA results from supernatants of control or LV-LTR
80/+60-
SaCas9-MOGS-treated primary CD4+ T cells of ART-suppressed patient living with HIV (PLWH). CD4+ T cells were isolated from frozen PBMCs and were stimulated with
anti-CD3 and anti-CD28 antibodies to stimulate latent HIV-1 for three days. Next cells were transduced with lentivirus expressing CRISPR-LTR
80/+60-SaCas9-MOGS and
viral load was assessed after 12 days. (F) qRT-PCR results showing the mRNA expression levels of HIV-1 Tat and (G) SaCas9. (H) Light microscopy images of control (top) or
LV-LTR
80/+60-SaCas9-MOGS-treated (bottom) primary CD4+ T cells (100). (I) ddPCR quantification of intracellular viral DNA in control and LV- LTR
80/+60-SaCas9-
MOGS-treated PWLH-derived CD4+ T cells. (J) ddPCR quantification of intracellular viral DNA in the HutR5 cells treated with supernatants derived cells in E. (K) Immunoblot
analysis of HIV-1 gp160, SaCas9-HA, and MOGS protein expression in control and LV-LTR
80/+60-SaCas9-MOGS-treated PWLH-derived CD4+ T cells. HIV-1 p55 and
a-tubulin were used as infection and loading controls. Unpaired t test (two-tailed) was used in (E), (F), and (J). **p < 0.01.

Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1017

 Molecular Therapy: Nucleic Acids

(legend on next page)

1018 Molecular Therapy: Nucleic Acids Vol. 32 June 2023

www.moleculartherapy.org

reduced level of infectivity in permissive cells (Figure 5C). As before,
no significant effect in viral replication was detected in the cells in a
primary infection (Figure 5B).
In the follow-up studies, using ex vivo cultures of primary CD4+ T cells
derived from virally controlled people with HIV-1 (PWH) provided
by the Comprehensive NeuroHIV Center (CNHC), we demonstrated
that Tat produced upon viral rebound induced CRISPR-MOGS
expression, ultimately resulting in the release of non-infectious virions
that limit the spread of the virus to uninfected cells (Figures 5E–5G).
Importantly, although in control-treated cells the virus-induced syn-
cytia (Figure 5H, top) were readily observed, they were completely ab-
sent in cells treated with Tat-inducible CRISPR-MOGS (Figure 5H,
bottom). Moreover, results from DNA assay showed significantly
lower levels of DNA per equal number of cells in CRISPR-MOGS-
treated cells than the control cells (Figure 5I). A combination of lower
level and infectivity of rebounded virus derived from Tat-inducible
CRISPR-MOGS-treated CD4+ T cells resulted in a drastic decrease
in the level of secondary infection compared with virus rebounded
from control cells (Figure 5J). Finally, results from the western blot
showed the overall decrease in the level of viral protein and more
importantly, the appearance of the larger size viral envelope protein
exhibiting SaCas9 reduced MOGS in the cells (Figure 5K).
Cooperative interaction of CRISPR-MOGS and CRISPR-HIV in
the elimination of HIV-1 infection
To investigate the combinatory effect of CRISPR designed for editing
of the MOGS gene for impeding viral entry along with the CRISPR en-
gineered for targeting and excising segments of the integrated proviral
DNA (CRISPR-HIV),30 the control or RNP-CRISPR-MOGS-treated
Jurkat cells were infected with HIV-1NL4-3-GFP-P2A-nef as before. Again,
CRISPR-MOGS treatment had no significant effect on the level of pri-
mary viral replication (Figures 6A and 6B), although the shift in the
migration of env glycoprotein was evident (Figure 6C). Next, superna-
tants from the control and the CRISPR-MOGS-treated primary in-
fected cells were used for the secondary infection of HutR5 cells fol-
lowed by the RNP treatment armed with CRISPR-HIV complex.
The CRISPR-HIV-mediated excision of the integrated proviral
DNA in HutR5 was verified using PCR genotyping. The appearance
of the 198 and 532 bp amplicons indicated the removal of the inter-
vening DNA fragments positioned between the 50 -LTR to Gag and
Gag to 30 -LTR, respectively (Figure 6D).30 The percent of the HutR5
infected cells (identified by GFP+ cells) and the level of viral load in
the media (determined by p24) along with the status of the viral enve-
lope protein (Figures 6E–6G), all together point to the functional co-
operativity between the CRISPR-mediated inactivation of MOGS and
the excision of HIV-1 proviral DNA in mitigating viral replication
during the secondary HIV-1 infection of human T lymphocytes.
Next, we adapted this strategy to ex vivo T cell cultures prepared from
the PWH (P02, P03, and P05) whose viral loads spontaneously re-
bounded. As shown in Figures 6H and 6I, a single treatment with
CRISPR-MOGS decreased the level of MOGS production along
with the appearance of larger glycoprotein and a decrease in the sec-
ondary viral infection. Lower levels of secondary infections were also
observed when rebound virus derived from CRISPR-HIV only treated
was used (Figure 6I). Importantly, combined treatments of the CD4+
T cells with CRISPR-MOGS and CRISPR-HIV-1 further declined,
albeit to various levels, the production of the infectious viral particles.
The different declines in viral levels between T cell cultures from
different donors might be the result of different viral loads and alter-
natively, variability in the efficiency of the delivery of CRISPR.
DISCUSSION
Mutations in the gene encoding MOGS cause the rare congenital dis-
order of glycosylation type II (CDG-II).31–33 Remarkably, people with
this disorder have no clinical evidence of reoccurring viral infections.
Cells derived from people with CDG-II show markedly reduced sus-
ceptibility to infection with glycosylation-dependent enveloped vi-
ruses like influenza and HIV-1 but not to non-enveloped viruses
(adenovirus, poliovirus) or glycosylation-independent enveloped vi-
ruses (vaccinia virus).28 Furthermore, iminosugars, glucose mimetic
glucosidase inhibitors, showed a broad spectrum antiviral activity
in vitro against multiple enveloped viruses like herpes, hepatitis b,
hepatitis c, west Nile, dengue Ebola, and coronaviruses including
SARS-CoV-2.34–37 Thus, the role of MOGS in refining the glycan is
suspected to be critical for optimal glycosylation of the viral envelope
protein and the morphogenesis of pathogenic HIV virions. Under this
assumption, glucosidase inhibitors (e.g., SC-48334, Zavesca, miglu-
stat, MDL 28,574A, celgosivir, Bu-CAST) have been evaluated in clin-
ical trials against HIV. The global application of pharmacologic inhib-
itors revealed unexpected, yet noticeable side effects including

Figure 6. A combination of CRISPR-MOGS and CRISPR-HIV treatments leads to improved virus clearance from in vitro infected T cell lines and primary
PWH-derived CD4+ T cells
(A) GFP-flow cytometry analysis of HIV-1NL4-3-GFP infection in WT and RNP-CRISPR-Cas9/MOGSgRNA-treated Jurkat cells. (B) HIV-1 gag p24 ELISA results for super-
natants collected from cells in (A). (C) Immunoblot analysis of HIV-1 gp160 and MOGS in the same cells. HIV-1 p55 and a-tubulin were used as infection and loading controls.
(D) PCR genotyping of CRISPR-Cas9-mediated 50 -LTR-gag and gag-30 -LTR excision of HIV-1 genome observed in HutR5 T-lymphoid cells infected with supernatants
derived from cells in (A) and electroporated with Cas9 only (lines 1 and 2) or Cas9/LTR1gRNA + Cas9/GagDgRNA RNP complexes (lines 3 and 4). For 50 LTR-Gag PCR, both
full-length 1,200 bp long and CRISPR-cleaved/end-joined 198-bp-long truncated amplicons are detected. For Gag-30 LTR PCR only CRISPR-cleaved/end-joined 532-bp-
long truncated amplicon is shown. (E) GFP-flow cytometry analysis of HIV-1NL4-3-GFP infection in the same cells. (F) HIV-1 gag p24 ELISA results for supernatants collected
from cells in (E). (G) Immunoblot analysis of HIV-1 gp160 and MOGS in the same cells. HIV-1 p55 and a-tubulin were used as infection and loading controls. (H) Immunoblot
analysis of HIV-1 gp160 and MOGS in untransfected, RNP-CRISPR-MOGS, RNP-CRISPR-LTR1/GagD, or RNP-CRISPR-MOGS+RNP-CRISPR-LTR1/GagD treated pri-
mary human CD4+ T cells isolated from PBMCs derived from three different people living with HIV (PLWH). HIV-1 p55 and a-tubulin were used as infection and loading
controls. (I) ddPCR analysis of viral DNA in the HutR5 cells incubated with supernatant as shown in (H) as a percentage of viral DNA copies detected in control cells. One-way
ANOVA and Dunnett’s multiple-comparisons test was used in (E) and (F). ****p < 0.0001.

Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1019

 Molecular Therapy: Nucleic Acids

Figure 7. Graphic illustration of CRISPR-mediated mitigation of HIV-1 infection by editing of MOGS and HIV-1 proviral DNA
(1) Uninfected cells. (2) Infection with HIV-1 followed by internalization and integration of viral DNA and replication of infectious virus that can be ceased upon treatment with
ART. (3) The treatment with a latency-reversing agent and/or ART removal results in reactivation of latent virus and production of Tat that potentiates CRISPR-MOGS and
CRISPR-HIV-1 that are sequentially delivered by transduction with Tat-inducible Cas9/MOGS gRNA and Cas9/HIV-1 gRNA LV or AAV. (4) Suppression of MOGS and
excision of segments of proviral DNA leads to defective proviral DNA (d-provirus). (5) Limited production of non-infectious HIV-1 due to aberrant pattern of env glycosylation
and defective viral genome.

intestinal distress and osmotic diarrhea,38–43 in part because of the
widespread application of the compound that non-specifically sup-
pressed host glucosidases. Regardless, the beneficial impact of the
path for the suppression of viral entry to host was deemed a powerful
strategy for antiviral therapy and required a more specific approach
toward the suppression of MOGS in the infected cells. As such, we
developed a genetic approach that is engineered to inactivate
MOGS gene expression by DNA editing that is exclusive to the im-
mune cells containing replication competent, latent HIV-1. We devel-
oped a functional pathway whereby activation of the silent virus and
the production of the viral protein, Tat, stimulate CRISPR gene edit-
ing apparatus that is delivered to the cells.29 This strategy is aimed at
editing of MOGS gene and is designed for the perturbation of glycan
configuration of the HIV-1 envelope protein that eventually results in
the production of non-infectious virions. Thus, it is reasonable to pre-
dict that, in a clinical setting, after the reactivation of latent proviral
DNA by latency-reversing agents (LRAs), and the expression of
Tat, when ART treatment is interrupted, the overall outcome will
be the appearance of non-infectious virus with no ability to spread af-
ter rebound from the reservoir. Indeed, the combination treatment
with CRISPR designed to excise a segment of proviral DNA, yet hav-
ing no effect on Tat production, further contributes to the elimination
of non-infectious viral particles. As noted, the strategy that is
described recruits the critically important HIV-1 protein, Tat, for
its replication to initiate a cascade of events that lead to the auto-elim-
ination of virus in the cells.
Importantly, limiting CRISPR gene editing to Tat-expressing and thus
HIV-1 infected cells provides an additional level of safety as it was re-
ported that CRISPR deletions are prone to induce p53-related pertur-
bations of cells,44,45 may impair cell viability and result in complex re-
arrangements of chromosomes.46,24 On a different, yet related note, it is
postulated that glycosylation of the Env protein functions as a shield to
protect the virus against immune-mediated cell defense. Thus, by inter-
fering with the formation of this barrier, newly replicated viral particles
may become more susceptible for serving as targets for elimination by
the immune cells.47,48 Therefore, it is plausible to speculate that non-in-
fectious viral progeny may offer a new pathway for the development of
a prophylactic vaccine from a weakened, immunogenic viral particles.
One may also speculate that the appearance of the inactive, non-infec-
tious viral particles may stimulate the immune system to overcome re-
maining infectious virus that may escape from elimination by CRISPR-
HIV. Thus, the outcome may eventually lead to permanent elimination
of HIV-1 in PWH and protects them from re-infection.
In conclusion, here, we propose a proof-of-principle design of a novel
and safe strategy that begins with the interruption of ART for control
of viremia followed by treatment with LRAs to stimulate the expres-
sion of CRISPR that is tailored for elimination of HIV-1 and inacti-
vation of MOGS (Figure 7).
MATERIALS AND METHODS
Cell culture
HEK293T cells (American Type Culture Collection, Manassas, VA)
and TZM-bl cells (obtained through the NIH HIV Reagent Program,
Division of AIDS, National Institute of Allergy and Infectious Diseases
[NIAID], NIH: TZM-bl cells, ARP-8129, contributed by Dr. John C.
Kappes and Dr. Xiaoyun Wu)15,16 were cultured in high-glucose

1020 Molecular Therapy: Nucleic Acids Vol. 32 June 2023

www.moleculartherapy.org
DMEM supplemented with 10% fetal bovine serum (FBS) and genta-
micin (10 mg/mL). Jurkat cells (clone E6, TIB-152; ATCC), HutR5 cells
(transformed human T cell line Hut78 stably transduced with CCR5, a
gift from Dr KewalRamani),49 U1 cells (obtained through the NIH
HIV Reagent Program, Division of AIDS, NIAID, NIH: anti-human
CD34 hybridoma [PR18], ARP-165, contributed by Dr. Thomas
Folks),20,50,51 J1.1 cells (obtained through the NIH HIV Reagent Pro-
gram, Division of AIDS, NIAID, NIH: HIV-1 lymphadenopathy-asso-
ciated virus [LAV]-infected Jurkat E6 cells [J1.1], ARP-1340, contrib-
uted by Dr. Thomas Folks),20,21 and ACH-2 cells (obtained through
the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH:
ACH-2 cells, ARP-349, contributed by Dr. Thomas Folks)20,22 were
cultured in RPMI medium containing 10% FBS and gentamicin
(10 mg/mL) (Sigma-Aldrich, St. Louis, MO). Peripheral blood mono-
nuclear cells (PBMCs) were isolated from whole blood by gradient
centrifugation on Ficoll-Paque for 30 min at 600  g followed by isola-
tion of CD4+ T cells using EasySep Human CD4+ T Cell Isolation Kit
(STEMCELL Technologies, Seattle, WA) and then were stimulated
with ImmunoCult Human CD3/CD28 T cell activator (STEMCELL
Technologies) for 3 days in RPMI with 10% FBS and gentamicin
(10 mg/mL) supplemented with human recombinant interleukin-2
(rIL-2) at a concentration of 30 ng/mL (STEMCELL Technologies).
Fresh media were exchanged every 2–3 days.
Design of gRNAs and construction of CRISPR-SaCas9-MOGS-
A/MOGS-B expression plasmid
The Benchling CRISPR guides designer tool (https://www.benchling.
com) was used to screen sequence of human MOGS (GeneBank
NG_008922.1) gene for possible gRNA protospacer regions followed
by SauCas9 specific NNGRR(N) PAM. A pair of gRNAs was selected
to introduce an 1,187 bp deletion in the coding sequence of the MOGS
gene (Figure S2A). Next, a pair of oligonucleotides with 50 -CACC and
30 -AAAC Bsa1 overhangs was obtained from Integrated DNA Tech-
nologies (Coralville, IA), annealed, phosphorylated, and ligated into
BsaI digested, dephosphorylated pX601-AAV-CMV: NLS-SauCas9-
NLS-3xHA-bGHpA; U6::BsaI-single guide RNA (sgRNA) (a gift
from Feng Zhang via Addgene, plasmid #61591). For multiplex
gRNA cloning, the target pX601-SauCas9-MOGS-B vector was line-
arized with EcoRI and KpnI, and the insertion fragment (U6-MOGS-
A-RNA scaffold) was produced via PCR using the primer pair T795/
T796 with a mutation of the 30 -end KpnI site (for further addition of
new sgRNAs) and the pX601-SauCas9-MOGS-A vector as the tem-
plate. After purification, the linearized vector and the insertion PCR
product were ligated using an In-Fusion HD Cloning Kit (Takara
Bio USA, San Jose, CA). The positive duplex sgRNA-expressing
pX601-SauCas9-MOGS-A+MOGS-B clones were identified by dou-
ble digestion with NotI and BamHI or EcoRI and verified using
Sanger sequencing. The same strategy was used to clone a single
MOGST4 gRNA into pX601 vector.
Generation the MOGS– single-cell clones
TZM-bl cells were co-transfected with pX601-MOGS-AB and pKLV-
U6gRNA-EF(Bbs1)-PGKpuro2ABFP (Addgene 62348, to provide pu-
romycin selection marker). Next day puromycin (1 mg/mL) was added
and cells were grown for a week. Surviving cells were plated in a 96-well
plate at a density of 1 cell/well/200 mL of growth medium and clonally
expanded for 3 weeks. Finally, expanded clones were screened using
PCR-genotyping for the excision of exons 1 and 2 of MOGS gene. Ju-
rkat MOGS
mixed cell population was purchased from Synthego
(Redwood City, CA) and clonally expanded in a round-bottom
96-well plates for 3 weeks. Next, expanded clones were screened using
PCR genotyping for the presence of indel mutations in exon 2 of
MOGS gene. U1, J1.1, ACH1, and HEK293T cells were electroporated
with ribonucleoprotein complexes composed of recombinant SaCas9
(Aldevron, Fargo, ND) and synthetic MOGST4 gRNA (Synthego). Af-
ter two days, transfected cells were clonally expanded and screened as
described above for Jurkat cells.
In vitro HIV-1 infections
HEK293T cells were transfected using CaPO4 precipitation method in
the presence of chloroquine (50 mM) with 25 mg of pNL4-3-GFP-
P2A-Nef29 or pNL4-3-BAL-GFP GFP (a gift from Dr. Christopher
Aiken, Vanderbilt University Medical Center, Nashville, TN)
2.5  106 cells/100 mm dish. The next day, medium was replaced,
and 24 and 48 h later, supernatants were collected, clarified at
3,000 rpm for 10 min, filtered through a 0.45 mm filter, and concen-
trated using ultracentrifugation for 2 h with 20% sucrose cushion.
Viral pellets were resuspended in Hank’s basic salt solution (HBSS)
by gentle agitation overnight and aliquoted. Viral titers were
measured using GFP-flow cytometry in HutR5 cells. For primary in-
fections, target cells were incubated with HIV-1NL4-3-GFP-P2A-Nef or
HIV-1NL4-3-BAL-GFP reporter virus at a multiplicity of infection
(MOI) of 0.25 for 4 h in growth medium then inoculum was removed,
cells washed twice with PBS and resuspended in growth medium. For
secondary infection HutR5 cells were incubated with supernatants
from primary infections overnight, next cells were washed twice
with PBS and resuspended in fresh growth medium.
RNP-CRISPR electroporations
Guide RNAs were coupled individually with SaCas9 by adding 2.2 mL
of sNLS-SaCas9-sNLS Nuclease (10 mg/mL = 80 mM; Aldevron) to
6 mL of synthetic gRNA (60 mM, CRISPRevolution Custom RNA
[modified], Synthego) in 11.8 mL of electroporation Buffer R (Neon;
Life Technologies, Carlsbad, CA, USA). The molar ratio when incu-
bating was 1:2 (9 mM:18 mM) SaCas9:gRNAs. The mixtures were
incubated individually for 10 min at room temperature (RT), then
combined and added to 2.5  106 of target cells in 200 mL Buffer R.
Cells were then mixed gently and electroporated using the Neon Elec-
troporation Device set to 1,400 V, 1  30 ms impulse with 100 mL
Neon tip. Cells were immediately plated in 4 mL of pre-warmed
growth medium.
Nucleic acid extractions and standard PCR and RT-PCR assays
Genomic DNA was extracted from cells using NucleoSpin Tissue kit
(Macherey-Nagel Inc., Bethlehem, PA). Briefly, the cell pellet was re-
suspended in 200 mL T1 followed by adding of 25 mL Proteinase K so-
lution (2.5 mg/mL) and overnight incubation at 55 C. The next day,
20 mL RNAse A (10 mg/mL) was added, and samples were incubated
Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1021

for 5 min at RT. The extraction was then completed according to the
manufacturer’s instructions. Genomic DNA was eluted in 100 mL
elution buffer (5 mM Tris/HCl [pH 8.5]) and quantified using Nano-
drop. For standard PCRs (Table S2), 250 ng extracted DNA was sub-
jected to PCR using Terra PCR Direct polymerase mix (Takara Bio)
under the following PCR conditions: 98 C for 2 min, 30 cycles
(98 C for 10 s, 60 C for 15 s, and 68 C for 120 s), 68 C for 7 min using
1st-round primers followed by nested PCR using diluted 1st-round
PCR. Next, amplicons extracted from the agarose gels were sent for
Sanger sequencing (Azenta, South Plainfield, NJ). For RT PCR, Mon-
arch Total RNA Miniprep kit (New England BioLabs Inc., Ipswich,
MA) was used for RNA extraction and Protoscript II First Strand
cDNA Synthesis Kit for reverse transcription. For gRNA expression
screening specific reverse primer (pX601gRNA scaffold/R;
Table S1) was used in RT reaction followed by standard PCR using
target LTR or Gag sense oligos as forward primers (Table S1) and
agarose gel electrophoresis. For checking saCas9 mRNA expression
oligo-dT primer mix was used in RT and cDNA was subjected to
PCR using saCas9 specific primer pairs and b-actin as a reference
(Table S1). Sanger sequencing results were analyzed using Clustal
Omega (EMBL-EBI) multiple sequence alignment tool and Sequence
Scanner Software 2 (Applied Biosystems).
ddPCR for quantification of HIV-1 DNA
ddPCR was performed on the basis of the water-oil emulsion droplet
technology, using the ddPCR Supermix for Probes reagents in the
QX200 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules,
CA). For quantification of HIV-1 DNA, the eluted cellular DNA was
PCR amplified using Taqman sets targeting HIV-c and as a reference
human TERT gene (Table S1). A total of 50–100 ng DNA was used as
template for ddPCR amplifications with thermal cycling conditions
used 98 C for 5 min, 45 cycles (98 C for 15 s, 60 C for 30 s). Data
acquisition and analysis were done using QX200 droplet reader and
QuantaSoft software provided with the instrument.
Antibodies and western blot
One million cells were lysed with RIPA buffer (25 mM Trizma base
[pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%
SDS) containing a protease inhibitor cocktail (Sigma-Aldrich) and
electrophoresed on 8% or 10% polyacrylamide SDS-PAGE gels using
1X Tris-Glycine-SDS running buffer. Gels were transferred onto
0.22 mm Odyssey nitrocellulose membranes (LI-COR, P/N 926-
31092) using the Bio-Rad Trans-Blot Turbo Transfer System, blocked
for 1 h in 5% milk + Tris-buffered saline-Tween 20 (TBS-T). Mem-
branes were probed with primary antibody in appropriate blocking
buffer overnight at 4 C. Membranes were washed three times with
TBS-T for 10 min at RT and probed with secondary antibody in
the same blocking buffer used for primary antibody at 1:10,000 dilu-
tion for 60 min at RT. Blots were washed as before. Images were ac-
quired using the Odyssey CLx Imaging System (LI-COR). Antibodies
used were glucosidase 1 (C11) (sc-374006; Santa Cruz Biotechnology,
Dallas, TX), gp160 (13390; NIH AIDS Reagent Program), a-tubulin
(32-2500; Life Technologies), HIV (3957; NIH AIDS Reagent Pro-
gram), HIV-1 gp120 (2343; NIH AIDS Reagent Program), SaCas9
1022 Molecular Therapy: Nucleic Acids Vol. 32 June 2023
Molecular Therapy: Nucleic Acids
(A01951; GenScript, Piscataway, NJ). For PNGnase F or Endo H
treatment, denatured protein lysates were incubated for 1 h at 37 C
with 500 units PNGase F or Endo H (New England BioLabs Inc.)
prior to acrylamide gel electrophoresis.
Cell proliferation, viability, cell cycle, and apoptosis assays
Jurkat and U1 WT and MOGS
cells were plated at the density of
1  105 cells/mL. An aliquot of cells was stained with propidium io-
dide (2 mg/mL) for 1 min for four consecutive days and analyzed using
Guava MiniCyte 8 flow cytometer (Luminex, Austin, TX). For the cell
cycle analysis 106 cells were fixed with 70% ethanol (cells were resus-
pended in 1 mL of 1 PBS and then dropwise added into 4 mL cold
88% ethanol) and stored at 4 C. The next day, fixed cells were spun
down, washed twice with PBS, and resuspended in staining buffer
containing propidium iodide (20 mg/mL) and RNAse A (200 mg/
mL) for 30 min at 37 C and then placed on ice and analyzed using
Guava MiniCyte 8 flow cytometer. Apoptotic assay was performed
using Guava Nexin Reagent (Luminex). Briefly, 105 cells in 100 mL
(1 PBS, 1% FBS) were mixed with 100 mL Guava Nexin Reagent
and incubated for 20 min at RT in the dark and then analyzed using
Guava MiniCyte 8 flow cytometer.
Construction of pPapi-LTR-SaCas9_MOGST4 plasmid
The U6-MOGST4 guide RNA expression cassette was sub-cloned
from pX601-MOGST4 plasmid into pPapi (a gift from John Doench
and David Root via Addgene plasmid #96921) vector by PCR using
Acc651-U6 F and Scaffold-BstBI R primers (Table S2) resulting in
pPapi-MOGST4 plasmid. Next, the original EF 1a promoter was re-
placed with the LTR (
80/+66) promoter using NheI/XbaI and
AgeI restriction sites. The final construct was named as pPapi-LTR-
SaCas9_MOGST4.
Electron microscopy
HutR5 T cells were incubated for 1 h with equal titers of virus-con-
taining supernatants collected from MOGS+ or MOGS
J1.1 cells
(300 ng Gag p24/107 cells). After 1 h, cells were spun down and fixed
in 2% glutaraldehyde for 1 h at RT (107 cells/10 mL). Next, cells were
treated with OsO4, stained with 0.5% uranyl acetate, pelleted in 2%
agarose, dehydrated in a dilution series of acetone/water, and
embedded in Spur’s resin (Electron Microscopy Sciences). The sec-
tions were examined with a JEOL JEM1010 transmission electron mi-
croscope (TEM) fitted with a side-mounted AMT XR-50 5Mpx CCD
camera. Whole-cell images were taken with 2,100 direct magnifica-
tion. Cell membrane and cytosolic regions were imaged with 15,000
direct magnification.
MALDI-glycan imaging
Virus-containing supernatants from ACH-2 (WT, MOGS
C17 and
C20) and J1.1 (WT, MOGS
C8, and C12) induced with TNF-a
(50 ng/mL) overnight were ultracentrifuged (25,000  g, 2 h, 4 C,
20% sucrose cushion), and the viral pellets were resuspended in
Urea Lysis Buffer (9 M urea, 50 mM Tris [pH 8], 100 U/mL Thermo
Universal Nuclease; Life Technologies). Purified viruses were spotted
on nitrocellulose as we have done with purified protein previously.25
www.moleculartherapy.org
Cell-based N-glycan imaging was adapted from previously published
protocols.26,27,52 J1.1 and U1 cells suspended in PBS were plated over
chambered hydrogel-coated slides (Nexterion Slide H) and incubated
for 1 h, allowing a monolayer of cells to covalently bind. The excess
cell suspension was removed, and the slide dried in a desiccator.
Bound cells were fixed with 10% neutral buffered formalin for
20 min. Cell chambers were removed, and cells were washed with
Carnoy’s solution (10% glacial acetic acid, 30% chloroform, 60%
ethanol) (10 min  2), 100% ethanol (2 min  2), and high-perfor-
mance liquid chromatography (HPLC)-grade water (3 min  2).
PNGase F PRIME (0.1 mg/mL in HPLC water) was sprayed onto the
slide using an M5 TM-Sprayer (HTX Technologies) for 10 passes at
25 mL/min, 1,200 mm/min, 45 C, 3 mm spacing between passes
with 10 psi nitrogen gas. The slide was incubated for 2 h at 37 C in
a humidity chamber. MALDI matrix a-cyano-4-hydroxycinnamic
acid (CHCA; 7 mg/mL in 50% acetonitrile/49.9% water/0.1% tri-
fluoroacetic acid) was applied using the same M5 TM- Sprayer for
10 passes at 70 mL/min, 1,300 mm/min, 79 C, 2.5 mm spacing be-
tween passes with 10 psi nitrogen gas. Ammonium phosphate mono-
basic (5 mM) was applied after matrix application for two passes at
70 mL/min, 1300 mm/min, 60 C, and 3 mm spacing between passes
with 10 psi nitrogen gas. A dual-source timsTOF flex MALDI-
QTOF mass spectrometer (Bruker) was used to conduct N-glycan
analysis. Cells were imaged using a SmartBeam 3D laser, operating
at 10,000 Hz using an M5 small smart beam at a 100 mm laser spot
size. Images were taken at a 150 mm raster with 600 laser shots per
pixel. Cells were analyzed with an m/z range of 700–4,000 in positive
ion mode. SCiLS Lab 2022a (Bruker) was used to analyze the data
generated. Mass spectra were normalized to total ion count.
b-lactamase viral entry assay
To generate HIV-1 virions containing active b-lactamase for use in
viral entry assays the pMM310 vector (obtained through the NIH
HIV Reagent Program, Division of AIDS, NIAID, NIH: HIV-1
YU2 Vpr b-lactamase expression vector [pMM310], ARP-11444,
contributed by Dr. Michael Miller [Merck Research Laboratories])23
was co-transfected into HEK293T WT or MOGS
(Clone M4) cells
together with pNL4-3 HIV-1 genome containing plasmids. After 48 h
the supernatants containing viral particles packaged with Vpr-b-lac-
tamase fusion protein were harvested and tittered by Gag p24 ELISA.
Next, TZM-bl cells were plated in 384-well plates at density
2  104cells/well and incubated with equal titers of HIV-1 virions
containing active b-lactamase (4 ng Gag p24/well). After 2 h CFF2-
AM substrate (Life Technologies) to final concentration of 1 ng/mL
was added and incubated for another 2 h. Then cells were fixed
with 2% paraformaldehyde (20 min) and analyzed using high content
image analysis. Viral entry was quantified in a confocal Operetta CLS
from PerkinElmer. Acquisition data from the 384 well plate was per-
formed using a 40 water immersion objective. Twenty fields were
acquired per well and three planes per field, corresponding to one-
quarter of the well. Total cells stained with CCF2-AM were detected
using the green channel (488/555 nm), and viral fusion-positive cells
were detected using the blue channel (400/430 nm). Data analysis was
performed using the Harmony 4.8 software from PerkinElmer. Spe-
cific parameters for total cell identification and viral infection were es-
tablished using a positive control (TZM-bl-pMM310) (Figure S9).
Positive viral entry cells were calculated using the maximum intensity
signal per cell, both TZM-bl-HIV BaL WT and TZM-bl-HIV BaL M4
were normalized against the signal from the control (TZM-bl-Untr).
The percentage of viral entry was calculated as the number of cleaved-
CCF2-AM-positive cells (blue) in each condition out of the total
number of cells identified per well (green, uncleaved-CCF2-AM-pos-
itive) (Figure 3B).
LV vector production
Lipofectamine 3000 (Life Technologies) was used for lentiviral pro-
duction. Specifically, HEK293T cells at about 75% confluency were
transfected in 100 mm cell culture plates with 3 mg pCMV-VSV-G
(a gift from Bob Weinberg via Addgene, plasmid #8484), 6 mg
psPAX2 (a gift from Didier Trono via Addgene, plasmid #12260)
and 8.5 mg pPapi-LTR-SaCas9_MOGST4 according to manufacturer
protocol. First viral supernatant was collected 24 h post-transfection,
which was combined with second viral supernatant collected about
52 h post-transfection. The combined viral supernatants were first
centrifuged at 2000 rpm to remove cell debris, then syringe filtered
through a 0.45 mm filter. After the clean-up, lentivirus was aliquoted
and stored in a
80 C freezer until use. The lack of recombination
between lentiviral vector truncated 50 -LTR/3’0 -LTRs and HIV-1
LTR
80/+60 Tat-inducible promoter in pPapi-LTR-SaCas9_-
MOGST4 vector was verified after packaging and after transduction
using qRT-PCRs and ddPCR, assays respectively (Figure S14).
Statistical analysis
Data were analyzed using Prism 9.0 (GraphPad, La Jolla, CA) and are
presented as mean ± SEM. Experiments were performed using a min-
imum of two biologically distinct replicates. One-way ANOVA and
Dunnett’s multiple comparisons test were used for statistical analyses.
For comparisons of two groups, a two-tailed unpaired t test was used.
DATA AVAILABILITY
The data supporting the findings of this study are available from the
corresponding author upon reasonable request. Source data for all fig-
ures will be provided upon request.
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.
1016/j.omtn.2023.04.027.
ACKNOWLEDGMENTS
The authors wish to thank past and present members of the Center for
Neurovirology and Gene Editing for their support and sharing of re-
agents. We express our gratitude to Dr. Gyorgy Csordas and Timothy
Schneider from Electron Microscopy Service Facility at MitoCare
Center for Mitochondrial Imaging Research and Diagnostics Depart-
ment of Pathology, Anatomy and Cell Biology, Thomas Jefferson
University, for preparation and imaging samples for TEM. We ex-
press our gratitude to Dr. Ilker Sariyer for his advice and valuable sug-
gestions during the course of this study and preparation of this
Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1023

manuscript. We appreciate Cynthia Papaleo for the assembly of data
and preparation of this manuscript. This work was made possible by
grants awarded by the NIH to K.K. and T.H.B. (P30MH092177) and
in part by W. W. Smith Charitable Trust to R.K. (grant A-1902). This
research was supported (in part) by the CRISPR for Cure Martin De-
laney Collaboratory for HIV cure UM1 AI164568-02 and co-funded
by NIAID, the National Institute of Mental Health (NIMH), the Na-
tional Institute on Drug Abuse (NIDA), the National Institute of
Neurological Disorders and Stroke (NINDS), the National Institute
of Diabetes and Digestive and Kidney Diseases (NIDDK), and the Na-
tional Heart, Lung, and Blood Institute (NHLBI).
AUTHOR CONTRIBUTIONS
K.K., R.K., C.R.Y.C., and C.M.B. conceived the idea and conducted
direct experiments. H.L., C.C., S.L., D.K.S., R.K., A.M., M.F., C.B.,
S.G., and J.D. conducted and performed experiments. K.K., T.H.B.,
R.K., C.C., T.J.C., J.G., A.M., C.M.B., and C.B. analyzed data. K.K.,
R.K., T.H.B., S.A., C.C., and C.M.B. prepared, commented on, and
edited the manuscript.
DECLARATION OF INTERESTS
K.K. and R.K. from Temple University and C.M.B. and C.R.Y.C. from
Children’s National Health System are named inventors on patents
that cover the viral gene editing technology for MOGS that is the sub-
ject of this article. C.M.B. and C.R.Y.C. hold patent 9,885,021 related
to MOGS editing of HIV-specific T cell therapies licensed by Mana
Therapeutics. C.M.B. and C.R.Y.C. are scientific cofounders of
Mana Therapeutics and serve on its scientific advisory board (SAB).
In addition to the foregoing interests, K.K. is a co-founder, board
member (observer), and chief scientific adviser and holds equity in
Excision Biotherapeutics, a biotech startup that has licensed the viral
gene editing technology from Temple University for commercial
development and clinical trials. T.H.B. holds equity in Excision Bio-
therapeutics and is a member of its SAB. T.J.C. and J.G. are members
of Excision Biotherapeutics, who provided advice and assisted in the
design of some part of experimental quality control of the gRNAs. All
other authors have no interests to disclose.
REFERENCES
1. Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson,
W.A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with
the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659.
2. Ray, N., and Doms, R.W. (2006). HIV-1 coreceptors and their inhibitors. Curr. Top.
Microbiol. Immunol. 303, 97–120.
3. Sattentau, Q.J. (1992). CD4 activation of HIV fusion. Int. J. Cell Clon. 10, 323–332.
4. Chen, B. (2019). Molecular mechanism of HIV-1 entry. Trends Microbiol. 27,
878–891.
5. Binley, J.M., Ban, Y.E.A., Crooks, E.T., Eggink, D., Osawa, K., Schief, W.R., and
Sanders, R.W. (2010). Role of complex carbohydrates in human immunodeficiency
virus type 1 infection and resistance to antibody neutralization. J. Virol. 84,
5637–5655.
6. Blough, H.A., Pauwels, R., De Clercq, E., Cogniaux, J., Sprecher-Goldberger, S., and
Thiry, L. (1986). Glycosylation inhibitors block the expression of LAV/HTLV-III
(HIV) glycoproteins. Biochem. Biophys. Res. Commun. 141, 33–38.
7. Coss, K.P., Vasiljevic, S., Pritchard, L.K., Krumm, S.A., Glaze, M., Madzorera, S.,
Moore, P.L., Crispin, M., and Doores, K.J. (2016). HIV-1 glycan density drives the
1024 Molecular Therapy: Nucleic Acids Vol. 32 June 2023
Molecular Therapy: Nucleic Acids
persistence of the mannose patch within an infected individual. J. Virol. 90, 11132–
11144.
8. Fischer, P.B., Collin, M., Karlsson, G.B., James, W., Butters, T.D., Davis, S.J., Gordon,
S., Dwek, R.A., and Platt, F.M. (1995). The alpha-glucosidase inhibitor
N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level
of post-CD4 binding. J. Virol. 69, 5791–5797.
9. Behrens, A.J., Vasiljevic, S., Pritchard, L.K., Harvey, D.J., Andev, R.S., Krumm, S.A.,
Struwe, W.B., Cupo, A., Kumar, A., Zitzmann, N., et al. (2016). Composition and
antigenic effects of individual glycan sites of a trimeric HIV-1 envelope g lycoprotein.
Cell Rep. 14, 2695–2706.
10. Cao, L., Pauthner, M., Andrabi, R., Rantalainen, K., Berndsen, Z., Diedrich, J.K.,
Menis, S., Sok, D., Bastidas, R., Park, S.K.R., et al. (2018). Differential processing of
HIV envelope glycans on the virus and soluble recombinant trimer. Nat. Commun.
9, 3693.
11. Crispin, M., Ward, A.B., and Wilson, I.A. (2018). Structure and immune recognition
of the HIV glycan shield. Annu. Rev. Biophys. 47, 499–523.
12. Struwe, W.B., Chertova, E., Allen, J.D., Seabright, G.E., Watanabe, Y., Harvey, D.J.,
Medina-Ramirez, M., Roser, J.D., Smith, R., Westcott, D., et al. (2018). Site-specific
glycosylation of virion-derived HIV-1 Env is mimicked by a soluble trimeric immu-
nogen. Cell Rep. 24, 1958–1966.e5.
13. Helenius, A., and Aebi, M. (2001). Intracellular functions of N-linked glycans. Science
291, 2364–2369.
14. Reily, C., Stewart, T.J., Renfrow, M.B., and Novak, J. (2019). Glycosylation in health
and disease. Nat. Rev. Nephrol. 15, 346–366.
15. Platt, E.J., Wehrly, K., Kuhmann, S.E., Chesebro, B., and Kabat, D. (1998). Effects of
CCR5 and CD4 cell surface concentrations on infections by macrophagetropic iso-
lates of human immunodeficiency virus type 1. J. Virol. 72, 2855–2864.
16. Wei, X., Decker, J.M., Liu, H., Zhang, Z., Arani, R.B., Kilby, J.M., Saag, M.S., Wu, X.,
Shaw, G.M., and Kappes, J.C. (2002). Emergence of resistant human immunodefi-
ciency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
Antimicrob. Agents Chemother. 46, 1896–1905.
17. Murrary, S.M., Zhang, Y., Douek, D.C., and Sekaly, R.P. (2020). Myeloid cells en-
riched for dendritic cell popultion from people living with HIV have altered gene
expression not restored by antiretroviral therapy. Front. Immunol. 11, 261.
18. Rappaport, J., and Volsky, D.J. (2015). Role of the macrophage in HIV-associated
neurocognitive disorders and other comorvidities in patients on effective antiretrovi-
ral treatment. J. Neurovirol. 21, 235–241.
19. Cohn, L.B., Chomont, N., and Deeks, S.G. (2020). The Biology of the HIV-1 latent
reservoir and implications for cure strategies. Cell Host Microbe 27, 519–530.
20. Symons, J., Chopra, A., Malatinkova, E., De Spiegelaere, W., Leary, S., Cooper, D.,
Abana, C.O., Rhodes, A., Rezaei, S.D., Vandekerckhove, L., et al. (2017). HIV integra-
tion sites in latently infected cell lines: evidence of ongoing replication. Retrovirology
14, 2. Erratum in: Retrovirology.
21. Perez, V.L., Rowe, T., Justement, J.S., Butera, S.T., June, C.H., and Folks, T.M. (1991).
An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization
after CD3 stimulation. J. Immunol. 147, 3145–3148.
22. Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P.,
Kovacs, J., Fauci, A.S., and Folks, T.M. (1989). Monokine regulation of human immu-
nodeficiency virus-1 expression in a chronically infected human T cell clone.
J. Immunol. 142, 431–438.
23. Cavrois, M., De Noronha, C., and Greene, W.C. (2002). A sensitive and specific
enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat.
Biotechnol. 20, 1151–1154.
24. Boutin, J., Cappellen, D., Rosier, J., Amintas, S., Dabernat, S., Bedel, A., and Moreau-
Gaudry, F. (2022). ON-target adverse events of CRISPR-cas9 Nuclease: more chaotic
than expected. CRISPR J 5, 19–30. https://doi.org/10.1089/crispr.2021.0120.
25. Black, A.P., Liang, H., West, C.A., Wang, M., Herrera, H.P., Haab, B.B., Angel, P.M.,
Drake, R.R., and Mehta, A.S. (2019). A novel mass spectrometry platform for multi-
plexed N-glycoprotein biomarker discovery from patient biofluids by antibody panel
based N-glycan imaging. Anal. Chem. 91, 8429–8435.
www.moleculartherapy.org
26. Blaschke, C.R.K., Black, A.P., Mehta, A.S., Angel, P.M., and Drake, R.R. (2020). Rapid
N-Glycan Profiling of Serum and Plasma by a Novel slide-based imaging mass spec-
trometry workflow. J. Am. Soc. Mass Spectrom. 31, 2511–2520.
27. Powers, T.W., Jones, E.E., Betesh, L.R., Romano, P.R., Gao, P., Copland, J.A., Mehta,
A.S., and Drake, R.R. (2013). Matrix assisted laser desorption ionization imaging
mass spectrometry workflow for spatial profiling analysis of N-linked glycan expres-
sion in tissues. Anal. Chem. 85, 9799–9806.
28. Moore, S.E., and Spiro, R.G. (1990). Demonstration that Golgi endo-alpha-D-man-
nosidase provides a glucosidase-independent pathway for the formation of complex
N-linked oligosaccharides of glycoproteins. J. Biol. Chem. 265, 13104–13112.
29. Kaminski, R., Chen, Y., Salkind, J., Bella, R., Young, W.B., Ferrante, P., Karn, J.,
Malcolm, T., Hu, W., and Khalili, K. (2016). Negative feedback regulation of
HIV-1 by gene editing strategy. Sci. Rep. 6, 31527.
30. Dash, P.K., Kaminski, R., Bella, R., Su, H., Mathews, S., Ahooyi, T.M., Chen, C.,
Mancuso, P., Sariyer, R., Ferrante, P., et al. (2019). Sequential LASER ART and
CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nat.
Commun. 10, 2753. https://doi.org/10.1038/s41467-019-10366-y.
31. Chang, J., Block, T.M., and Guo, J.T. (2013). Antiviral therapies targeting host ER
alpha-glucosidases: current status and future directions. Antivir. Res. 99, 251–260.
32. Grunewald, S., Matthijs, G., and Jaeken, J. (2002). Congenital disorders of glycosyla-
tion: a review. Pediatr. Res. 52, 618–624.
33. Sadat, M.A., Moir, S., Chun, T.W., Lusso, P., Kaplan, G., Wolfe, L., Memoli, M.J., He,
M., Vega, H., Kim, L.J.Y., et al. (2014). Glycosylation, hypogammaglobulinemia, and
resistance to viral infections. N. Engl. J. Med. 370, 1615–1625.
34. Alonzi, D.S., Scott, K.A., Dwek, R.A., and Zitzmann, N. (2017). Iminosugar antivirals:
the therapeutic sweet spot. Biochem. Soc. Trans. 45, 571–582.
35. Casas-Sanchez, A., Romero-Ramirez, A., Hargreaves, E., Ellis, C.C., Grajeda, B.I.,
Estevao, I.L., Patterson, E.I., Hughes, G.L., Almeida, I.C., Zech, T., and Acosta-
Serrano, Á. (2021). Inhibition of protein N- glycosylation blocks SARS-CoV-2 infec-
tion. mBio 13, e0371821.
36. Chang, J., Block, T.M., and Guo, J.T. (2015). Viral resistance of MOGS-CDG patients
implies a broad- spectrum strategy against acute virus infections. Antivir. Ther. 20,
257–259.
37. Watanabe, Y., Bowden, T.A., Wilson, I.A., and Crispin, M. (2019). Exploitation of
glycosylation in enveloped virus pathobiology. Biochim. Biophys. Acta Gen. Subj.
1863, 1480–1497.
38. Fischl, M.A., Resnick, L., Coombs, R., Kremer, A.B., Pottage, J.C., Jr., Fass, R.J., Fife,
K.H., Powderly, W.G., Collier, A.C., Aspinall, R.L., et al. (1994). The safety and effi-
cacy of combination N-butyl- deoxynojirimycin (SC-48334) and zidovudine in pa-
tients with HIV-1 infection and 200-500 CD4 cells/mm3. J. Acquir. Immune Defic.
Syndr. 7, 139–147.
39. Hoechst, M.R. (1996). A randomized, double-blind active-controlled, dose-ranging
study of the safety and efficacy of chronically administered MDL 28,574A in the treat-
ment of HIV-infected patients. NLM identifier: NCT0002151.
40. Hoechst, M.R. (1996). A study of the safety and efficacy of chronically administered
MDL 28,574A in the treatment of HIV-infected patients. NLM identifier:
NCT0002150.
41. Searle, G.D. (1992). Initial Phase II Efficacy and Safety Study of SC-48334
Administered in Combination with Low-Dose Zidovudine (AZT) to Symptomatic
HIV-1 Infected Patients with = or > 200 to = or < 500 CD4+ Cells/mm3. NLM
Identifier: NCT0001993.
42. Searle, G.D. (1993). Phase II Study of the Safety and Surrogate Marker Efficacy of
Butyldeoxynojirimycin (SC-48334) and AZT in Symptomatic HIV-1 Infected
Patients with 200- 500 CD4+ Cells/mm3. NLM Identifier: NCT0002079.
43. Tierney, M., Pottage, J., Kessler, H., Fischl, M., Richman, D., Merigan, T., Powderly,
W., Smith, S., Karim, A., Sherman, J., et al. (1995). The tolerability and pharmacoki-
netics of N-butyl-deoxynojirimycin in patients with advanced HIV disease (ACTG
100). The AIDS clinical trials group (ACTG) of the national institute of allergy and
infectious diseases. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 10, 549–553.
44. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B., and Taipale, J. (2018). CRISPR-
Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24,
927–930. https://doi.org/10.1038/s41591-018-0049-z.
45. Enache, O.M., Rendo, V., Abdusamad, M., Lam, D., Davison, D., Pal, S., Currimjee,
N., Hess, J., Pantel, S., Nag, A., et al. (2020). Cas9 activates the p53 pathway and selects
for p53-inactivating mutations. Nat. Genet. 52, 662–668. https://doi.org/10.1038/
s41588-020-0623-4.
46. Turchiano, G., Andrieux, G., Klermund, J., Blattner, G., Pennucci, V., El Gaz, M.,
Monaco, G., Poddar, S., Mussolino, C., Cornu, T.I., et al. (2021). Quantitative evalu-
ation of chromosomal rearrangements in gene-edited human stem cells by CAST-
Seq. Cell Stem Cell 28, 1136–1147.e5. https://doi.org/10.1016/j.stem.2021.02.002.
47. He, S., and Wu, Y. (2019). Relationships between HIV-mediated chemokine corecep-
tor signaling, cofilin hyperactivation, viral tropism switch and HIV-mediated CD4
depletion. Curr. HIV Res. 17, 388–396.
48. Sun, L., Paschall, A.V., Middleton, D.R., Ishihara, M., Ozdilek, A., Wantuch, P.L.,
Aceil, J., Duke, J.A., LaBranche, C.C., Tiemeyer, M., and Avci, F.Y. (2020).
Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses
by eliciting T cell help. Nat. Commun. 11, 2550.
49. Wu, L., Martin, T.D., Vazeux, R., Unutmaz, D., and KewalRamani, V.N. (2002).
Functional evaluation of DC- SIGN monoclonal antibodies reveals DC-SIGN inter-
actions with ICAM-3 do not promote human immunodeficiency virus type 1 trans-
mission. J. Virol. 76, 5905–5914.
50. Hu, W., Kaminski, R., Yang, F., Zhang, Y., Cosentino, L., Li, F., Luo, B., Alvarez-
Carbonell, D., Garcia-Mesa, Y., Karn, J., et al. (2014). RNA-directed gene editing spe-
cifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci.
USA 111, 11461–11466.
51. Folks, T.M., Justement, J., Kinter, A., Dinarello, C.A., and Fauci, A.S. (1987).
Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell
line. Science 238, 800–802.
52. Angel, P.M., Saunders, J., Clift, C.L., White-Gilbertson, S., Voelkel-Johnson, C., Yeh,
E., Mehta, A., and Drake, R.R. (2019). A rapid array-based approach to N-Glycan
profiling of cultured cells. J. Proteome Res. 18, 3630–3639.
Molecular Therapy: Nucleic Acids Vol. 32 June 2023 1025
OMTN, Volume 32

Supplemental information

Strategic self-limiting production

of infectious HIV particles
by CRISPR in permissive cells
Hong Liu, Chen Chen, Shuren Liao, Danielle K. Sohaii, Conrad R.Y. Cruz, Tricia H.
Burdo, Thomas J. Cradick, Anand Mehta, Carlos Barrero, Magda Florez, Jennifer
Gordon, Stephane Grauzam, James Dressman, Shohreh Amini, Catherine M.
Bollard, Rafal Kaminski, and Kamel Khalili
 SUPPLEMENTAL FIGURES

Figure S1. N-linked protein glycosylation pathway and MOGS. (A) Schematic of early steps of

the N-glycosylation pathway in the ER lumen. Upon completion of the synthesis, the membrane-

bound precursor sugar chain (glycan) is composed of a total of 14 sugar residues

(Glc3Man9GlcNAc2) (1). Next, the precursor glycan is transferred from dolichylpyrophosphate

carrier (2) to an asparagine residue in the conserved motif Asn-X-Ser/Thr of the growing, nascent

polypeptide chain (3). MOGS is responsible for trimming the first glucose residue from the glycan

chains of nascent glycoproteins (4). This step is followed by further sugar trimming (5-6), which

continues in cis and medial Golgi compartments. The final addition of terminal sugar residues in

trans Golgi results in complex-type glycans (not shown). (B) In the presence of MOGS, the HIV-1

Env is decorated with a mosaic of glycans, including fully processed complex-type glycans (left). The

1
lack of MOGS results in a glycan trimming defect and the production of HIV-1 Env decorated with

only unprocessed precursor high-mannose-type glycans (right). Glycan shield composed of

unprocessed high-mannose-type glycans causes a shift in glycoprotein size which can be detected

by Western blot.

2
A

B C

D
E

Figure S2. Generation of CRISPR-based vector for disrupting MOGS expression. (A) The

schematic of the MOGS gene. The MOGS gene is located in chromosome 2 and comprises of

4326 nucleotides with four exons. The positions of the target sequences for gRNAs A and B are

shown by arrows. (B) Nucleotide sequence of the 5’ region of the MOGS gene. A pair of guide

RNAs are designed to target promoter and intron2 regions of the MOGS gene. Successful cleavage

at the target sites will lead to the deletion of the 1187bp long segment of DNA spanning exon1-

intron1-exon2, which contains the MOGS start codons. (C) The map of the pX601-MOGS-AB

plasmid. The “all-in-one" plasmid contains expression cassettes of Cas9 and two guide RNAs

targeting MOGS gene. (D) Agarose gel pictures showing results of RT-PCR. The pX601-MOGSAB

plasmid was transfected into TZM-bl cells. After 3 days and 6 days, the expression of SaCas9 and

guide RNAs MOGSA and B was confirmed by reverse-transcription PCR. (E) The excision of the

MOGS gene was detected by PCR. The arrows point to intact full- length 1620bp long MOGS

3
promoter-intron 2 amplicon (top arrow) and CRISPR-cleaved/end- joined 433bp truncated

amplicon (bottom arrow), carrying 1187 bp deletion of exon 1-2 region of the MOGS gene.

4
Figure S3. PCR DNA amplification of several potential cellular target genes for off-target

cleavage. Agarose gel analysis of PCR amplification of the top five bioinformatically nominated

off-target sites in human genome using genomic DNA obtained from wildtype TZM-bl cells and two

MOGS knockout clones.

5
Figure S4. Sanger DNA sequence tracing of the amplicons purified from the agarose gels. Analysis

was done by ICE software.

6
Figure S5. Cell growth and viability of the MOGS knockout Jurkat and U1 cells. (A-B) To

investigate the impact of MOGS gene knockout on cell growth, 1 x 105 to 2 x 105 cells/ml Jurkat

(Panel A) or U1(B) were seeded and after 4 days the average number of live and dead cells were

determined by staining with propidium iodide and quantified by Guava machine. (C-D) Graphs

show live Jurkat (C) and U1 (D) cell numbers over a period of 4 days as determined by trypan-blue

staining. Experiments were performed in triplicates.

7
Figure S6. Cell Cycle progression and apoptosis analysis of the MOGS knockout Jurkat and

U1 cells. (A-B) Apoptosis assay. Bar graphs show the average results of apoptotic assay carried

out on Wild-type and MOGS knockout cells. The experiments were performed in triplicates. Guava

Nexin Reagent was used to investigate apoptosis using 1X105 cell samples. Briefly, cells were

mixed with equal volume of Guava Nexin Reagent as suggested by the supplier. The fractions of

each apoptotic stages of cells were evaluated on Guava as instructed. The different colors of the

8
bar graphs indicate the average percentage of each apoptotic stages. (C-D) Illustration of the

results from Guava MiniCyte for assessing Cell Cycle progression. The percent of cells at each

stage were determined as specified by the guava manual. The average percentage of each cell

cycle stages was represented in different colors on the bar graphs. As seen, no significant

differences were observed between wild-type and MOGS knockout cells.

9
Figure S7. CRISPR-Cas9 mediated MOGS gene editing in primary CD4+ T cells. (A) Agarose

gel analysis of PCR amplification of the exon 2 of the MOGS gene. Four guide RNAs were designed

to target the exon two regions of the MOGS gene. Primary CD4+ T cells were electroporated with

RNA-protein complex (RNP) composed of synthetic gRNA and recombinant Cas9 protein and then

detected for the MOGS by gene-specific PCR. (B) MOGS gene knockout score analysis. The PCR

products from Panel A were purified from the gel and analyzed by Sanger sequencing. The

CRISPR-Cas9 induced knockout-score for specific indel mutations at the four gRNA target sites

was calculated using ICE (inference of CRISPR edits) tool (Synthego). (C) GFP-flow cytometry

analysis of primary CD4+ T cells infected with HIV-1-Bal- GFP. The percentage of GFP positive cells

were detected by FACS on day 2 after viral infection.

10
Figure S8.

11
Figure S8A (Figure 3 Sub-Panel A1)

12
Figure S8B (Figure 3, Sub-Panel A2)

13
Figure S8C (Figure 3, Sub-Panel A3)

14
Figure S8D (Figure 3, Sub-Panel A4)

15
Figure S8E (Figure 3, Sub-Panel B1)

16
Figure S8F (Figure 3, Sub-Panel B2)

17
Figure S8G (Figure 3, Sub-Panel B3)

18
Figure S8H (Figure 3, Sub-Panel B4)

19
Figure S8. Electron microscopy image of target HuTR5 cell surface after 1h incubation with virus-

containing supernatants harvested from J1.1 WT cells. Different stages of the attachment and entry

of the infectious virions into the target cell are seen.

20
Figure S9. High Content Image Analysis of -lactamase viral entry assay. Specific parameters

for cell identification (CCF2-AM-positive, green) (A) and viral entry (CCF2- positive, blue) (B) were

established, as shown using a positive control (TZM-bl-pMM310). Data analysis was performed

using the Harmony 4.8 software from PerkinElmer.

21
Figure S10. Immunoblot analysis of HIV-1 gp160 in untreated, PNGase F or Endo H digested

cellular lysates from PMA-SAHA stimulated U1 cells. HIV-1 p55 was used as controls.

22
Figure S 11. Increased levels of hyper-glucosylated glycans observed in MOGS K/O

lymphoid and myeloid cells via MALDI-glycan imaging of cells. (A) The level of the Glc3Man9

glycan in either wild type Jurkat cells (J), MOGS deleted Jurkat cells (JM), wild type U1 cells (U) or

MOGS deleted U1 cells (UM). Panels B-C show the level of the Glc3Man8 (B) and Glc3Man7 (C)

23
glycan in the same samples. The abundance key is shown for each panel, where blue is low

abundance and red is high abundance. (D-G) Deletion of MOGS leads to altered glycosylation.

24
Figure S12. Strategic activation of CRISPR-MOGS by HIV-1 Tat. (A) Immunoblot analysis of

SaCas9-HA in HEK293T cells transfected with pLTR-80/+60-SaCas9- MOGS, pLTR-120/+60-

SaCas9-MOGS, or pLTR-454/+60-SaCas9-MOGS constructs together with empty pCMV (Tat-) or

pCMV-Tat (Tat+) plasmids. -tubulin was used as a loading control. (B) Immunoblot analysis of

SaCas9-HA, gp160, HIV Gag p55 and housekeeping ɑ-tubulin in HIV-1NL4-3-GFP infected control or

pLTR-80/+60-SaCas9-MOGS +/- pCMV-Tat transfected HEK293T. HIV-1 Gag p55 and -tubulin

were used as infection and loading controls. (C) GFP-flow cytometry analysis of HIV-1NL4-3-GFP

infection in the same cells. (D) GFP-flow cytometry analysis of HutR5 T-lymphoid cells infected

with supernatants derived from cells in B). One-way ANOVA and Dunnett’s multiple comparisons

test was used in Panel D. p < 0.0001 and is indicated by ****.

25
Figure S 13. DNA structure of pPapi-LTR-SaCas9_MOGST4 was verified by sequencing.

26
Figure S14. The absence of recombination between lentiviral vector 3’LTR and HIV-1 LTR -

80/+60 Tat-inducible promoter. (A) Sequence alignment between the lentiviral vector 5’ and 3’ LTRs

and used in the study HIV-1 LTR -80/+60 Tat-inducible promoter. (B) Linear map of Tat-inducible

27
HIV-1 LTR -80/+60-SaCas9-MOGS and control EF-1-SaCas9-MOGS lentiviral vectors. The

positions of Taqman primer/probe sets used in the assay are highlighted in pink. The RRE probe

binds between lentiviral vector 5’LTR and SaCas9 promoter (LTR -80/+60 or EF-1) and the SaCas9

probe binds between SaCas9 promoter (LTR -80/+60 or EF-1) and lentiviral vector 3’LTR. Loss of

SaCas9 signal would indicate recombination between HIV-1 -80/+60 Tat-inducible promoter and

lentiviral vector 3’LTR. (C) Results of ddPCR assay using genomic DNA isolated from HEK293T cells

transduced with LV-LTR -80/+60-SaCas9-MOGST4 or LV-EF-1-SaCas9-MOGST4 lentiviral

vectors. No changes in RRE/SaCas9 signal ratio were detected in LV-LTR -80/+60-SaCas9-MOGST4

treated compared to LV-EF-1-SaCas9-MOGST4 treated cells indicating the lack of recombination

events between HIV-1 -80/+60 Tat-inducible promoter and lentiviral vector 3’LTR. Note that the

recombination between lentiviral vector 5’LTR and HIV-1 -80/+60 Tat-inducible promoter would result

in loss of the packaging signal (HIV-1 ψ, yellow) and production of empty lentiviral particles.

28
Table S1. Potential off target sites in human genome for MOGS target A and B

MOGSA sequence PAM On-target Score Off-target Score

AAAAACCAATCTAACGTTGGG CTGAG 22.9 93.5
Sequence PAM Score Gene Chromosome Mismatche On-target
d
AATAACCAATATAACGTTTGG AGGGA 0.5 chr1:-223007178 3 FALSE
TAAAACTCATCTAAAGTTGGG CAGAG 0.4 chr18:+75408839 4 FALSE
TTAAACCAACCTAAAGTTGGC AGGGA 0.1 chr7:-145084841 5 FALSE
TCAAATCAATCTAACTTTTGG ATGGA 0.1 chr2:-1252277 5 FALSE
AAACAGCAATCCAACGTTGGA CAGAA 0.1 chr15:+89058842 4 FALSE
ATAAACCAATGTAAAGTTTGG GGGGA 0.1 chr18:-77125281 4 FALSE
TAAAATAAATCTAACGTTTTG GAGAA 0.1 chr7:-39007024 5 FALSE
ATAAACCAATCTTACTTTAGG AAGGA 0.1 chrY:-6848825 4 FALSE
AAAAATAAATCTAAAGTTGGA GAGGG 0.1 chr5:-59064772 4 FALSE
TAAAATGAATCTAACTTTGAG TGGAA 0.1 chr4:-168055825 5 FALSE
TAAAAGCAATCTTACGTTTGT TTGAA 0.1 chr12:-45151581 5 FALSE
TAATACCAATTTAACGGAGGG TAGGG 0.1 chr3:-182494891 5 FALSE
CAAAATCAATCTAATGTAGGA AAGGA 0.1 chr2:-141710926 5 FALSE
ATAATCCACTCTAACTTTGGG CAGGA 0.1 chr11:+4179686 4 FALSE
TAAAACTAATATAAAGTTGGA AAGAA 0.1 chr3:-82582701 5 FALSE
GAAAACAAATATAAGGTTGGA TTGGG 0.1 chr5:+71583444 5 FALSE
ATTAACCAGTCTAAGGTTGGG ATGGA 0.1 chr1:-83708474 4 FALSE
AAAAACCTATGTAACGTTTAG GGGAA 0.1 chr1:-164679124 4 FALSE
GAAAACAAATCTAACCTTATG TGGAA 0.1 chr9:-113329284 5 FALSE
AAAAACTAATTTAACATTAGG TAGAA 0.1 chr10:+16939791 4 FALSE
AAAAATCCATCTAATGGTGGG TGGGA 0.1 chr4:-181429293 4 FALSE
AAATAACAAGCTAACTTTGGG AGGAG 0.1 chr3:-174640046 4 FALSE
TCAGGCCAATCTAATGTTGGG TGGAA 0.1 chr1:-209342435 5 FALSE
TAACATCAATCTGAAGTTGGG GTGAG 0.1 chr20:+20306629 5 FALSE
CAAGACCAATGTATCATTGGG AGGAG 0.1 chr6:-11666409 5 FALSE
AAATCCCAAGCTAAAGTTGGG AGGAG 0.1 chr6:+99270525 4 FALSE
CAAAACCCATCTACCCTTGCG CGGGG 0.1 chr17:-45161763 5 FALSE
CAAAACCAAAATAACATTGGA AGGAA 0.1 chr4:+138853014 5 FALSE
CACAACCATTATAAAGTTGGG CTGAG 0.1 chr3:-96829074 5 FALSE
GAAAACAAATCTAAAATTTGG CAGAA 0.1 chr2:-227631204 5 FALSE
AAAAACCCATCTGCCGTTGAG AGGAA 0.1 chr20:+55451376 4 FALSE
GAAAATCAATCTGATGGTGGG TCGGG 0.1 chr7:+45709300 5 FALSE
TAAAACCAACCTCAAGTTGGT GGGGA 0.1 chr6:-35202448 5 FALSE
CAAAACCAACCTAACTTGGGT TTGAG 0.1 chr4:-169047258 5 FALSE
CAACACCAAACCAAAGTTGGG TTGAA 0.1 chr2:+229585812 5 FALSE
GAAATCCAAGGTAACCTTGGG CTGAG 0.1 chr8:-57256433 5 FALSE
TAAAAGCAATGTAATATTGGG AGGGG 0.1 chr4:+165706353 5 FALSE
AAAAAAGAATTTAACTTTGGG TAGAA 0.1 chr4:+74769673 4 FALSE
AAAAAGTAATATAACATTGGG ATGAA 0.1 chr1:-66983188 4 FALSE
CTAAACTTATCTGACGTTGGG ATGAG 0.1 chr10:+21061613 5 FALSE
AAAAACCAAACTAATGTTCTG GGGGG 0.1 chr9:+97484825 4 FALSE
AAAAAGCAGTCTCACTTTGGG AGGAG 0.1 chr20:-757338 4 FALSE
AGACAGCAATCAAACGTTGGG AGGAG 0.1 chr17:+11928614 4 FALSE
AAAAAACCATGTAAGGTTGGG TGGGA 0.1 chr8:-94332742 4 FALSE
CAAAATAAAACTAATGTTGGG CTGGG 0.1 chr6:+46298368 5 FALSE
TAAAACCAATAAAAAGTTGAG GAGAA 0.1 chr1:-76468822 5 FALSE
AAAAACCAATTTACCTTTGAG GAGAG 0.1 chrX:-103732828 4 FALSE
TAAAACAAATACAAGGTTGGG ACGAA 0.1 chr9:+62855232 5 FALSE
TAAAACAAATACAAGGTTGGG ACGAA 0.1 chr9:+41290897 5 FALSE
GAAAACCAATCTCAGTTTGGA AAGGG 0.1 chr20:+43291205 5 FALSE

29
MOGSB sequence PAM On-target Score Off-target Score
TATACAGGCAGTTGTTTAAGG CAGGA 35.8 84.7
Sequence PAM Score Gene Chromosome Mismatche On-target
AAGACAGGCAGTTGTTTCAGA GAGAG 0.6 chr9:+109250597 4 FALSE
ATTACAGGCAGTTTTTTATGG TAGAG 0.5 chr1:+69318870 4 FALSE
GAGACAGGCAGTTGTTTAGTG GTGGA 0.5 chr5:-170555135 4 FALSE
TTTACAGGCAGCTGTTTTAGG AGGGG 0.5 chr7:-47773752 3 FALSE
AATTCAGGCAGGTGTTTAATG GAGAA 0.5 chr2:-140622360 4 FALSE
GAAAGAGGCAGTTGTTTTAGG ATGAG 0.5 chr9:+31766690 4 FALSE
AATACATGCATTTGTTTATGG AGGAA 0.4 chrY:-5814288 4 FALSE
GATAAAGGCAGTTGCTGAAGG AAGAG 0.4 chr2:-119219992 4 FALSE
AATACATGCATTTGTTTATGG AGGAA 0.4 chrX:-92697525 4 FALSE
CATACAGGAAGTTGATTAATG AGGAA 0.4 chr1:-147676835 4 FALSE
CATTTAGGCAGTTGCTTAAGG GTGAA 0.4 chr18:+61654239 4 FALSE
TATACAGGCAGCTGCTAAAGG CTGGA 0.3 chr11:-30396341 3 FALSE
TATACTGACAATTGTTTAAGG AAGAA 0.3 chr3:-159702981 3 FALSE
GATACAGGGTGTTTTTTAAGG TGGGG 0.3 chrX:-2661444 4 FALSE
GTTACAGGCATTTGTTCAAGA CAGAA 0.1 chr16:-60644045 5 FALSE
AGTACAGGCAGTTGATTAACC ACGAA 0.1 chr6:-116206219 5 FALSE
CAGACAAGCAGTTGTTAAATG TAGAG 0.1 chr7:+136704248 5 FALSE
AAAACAGGCAATTGTTTGAGA GAGAG 0.1 chr10:+26802472 5 FALSE
GCTGCAGGCAGTTGTATGAGG ATGAA 0.1 chr7:-27126806 5 FALSE
ATTACAGGCAATTATTTAAGA AAGAA 0.1 chr1:+90897534 5 FALSE
AATAAATGCAGTTGTTCAAGA TAGAA 0.1 chr3:-82792950 5 FALSE
CATGGAGGCAGATGTTTAAGA GAGGA 0.1 chr1:+5950069 5 FALSE
AATCCGGGCAGTTGTGTAAAG GAGAA 0.1 chr21:-36833896 5 FALSE
AATTCTGGCAGTGGTTTAAGC AAGAG 0.1 chr20:-42169459 5 FALSE
AATATAGGCATTTGTTTAAAT GGGAG 0.1 chr22:+30120221 5 FALSE
TAAACAGGCAATTGTTAAAAG AGGAA 0.1 PRDM6 chr5:+123192360 4 FALSE
AAAACAGGCAGCTGTTAAAAG GTGAA 0.1 chr8:+110123016 5 FALSE
AAGACAGGCAGTTGTGTGAGT ACGGA 0.1 chr8:-1700154 5 FALSE
GATAGAGACAGTTGTGTAAGA GAGGA 0.1 chr3:+45454832 5 FALSE
CAAACAGGAAGTTATTTAAAG CAGGG 0.1 chr12:+11596210 5 FALSE
AAGAGAGGCAGTTGTATTAGG CAGGA 0.1 chr2:+152602180 5 FALSE
AATTCAGGCAATTGTTTTAAG ATGAA 0.1 chr11:+13247842 5 FALSE
CATTGAGGGAGTTGTTTAAGC ATGAA 0.1 chr6:-155000183 5 FALSE
TTTAGAGGAAGTTGTTTAGGG ATGGG 0.1 chr4:+116739523 4 FALSE
CACACAGACAGTTGCTTAGGG CAGAA 0.1 chrX:-136218777 5 FALSE
AATAAAGGCAGTTCTTTAAAC CTGAG 0.1 chr14:-79731302 5 FALSE
GATATAGCCAGTTTTTTAAGT ACGAA 0.1 chr5:+159188976 5 FALSE
CATAAAGGCATTTGTTGAAGA TAGGA 0.1 chr13:+11323345 5 FALSE
GATATAGGGAGTTATTTAAGT CTGAG 0.1 chr17:-48531927 5 FALSE
AAGGCAGGCAGTTGGTCAAGG CAGAA 0.1 chr16:+19157660 5 FALSE
AATATAGGCAGTTCTTTGAGC AAGAA 0.1 chr11:-14710937 5 FALSE
CAAACACACAGTTGTTTAAAG GAGAA 0.1 chr6:+18902380 5 FALSE
TATTCAGGCAGTTGTAAAAGA CAGGA 0.1 chr13:+81836921 4 FALSE
TATAGAGGCAGTTGCTTATGT AAGGA 0.1 chr13:-65632940 4 FALSE
TATGCTGGGAGTTGTTTAAAG GAGAA 0.1 chr15:-91651756 4 FALSE
TGTTCAGGCTGTTGTTCAAGG CTGGA 0.1 chr2:-175748993 4 FALSE
TATACTGGCTGTTGCTTAAGA AAGAA 0.1 chr3:+83732988 4 FALSE
ATTACAGGCAGATCTTTGAGG TGGAG 0.1 chr13:+51261581 5 FALSE
CATGGAGGCAGGTGTTTTAGG CAGAA 0.1 chr10:+12939256 5 FALSE
TATAAAAGCAATTGTTTAATG AGGAA 0.1 chr21:+33525802 4 FALSE

30
Table S2.

PCR Primer name Sequence (5’→3')

1. Standard PCRs

MOGS-A/MOGS- B 1st round MOGS F GGATGTCGACAAATGCAACGGTAG

EXCISION
1st round MOGS R ACGGTGTTTCTCCATTTTGG

2nd round MOGS F TCCTCATGTCTGGTGATCCA

2nd round MOGS R CATTTTGGTCAGGCTGGTCT

5'LTR-gag LTR F TGGAAGGGTTAATTTACTCC

EXCISION
gag R ACCATTTGCCCCTGGAGGTT
Gag-3'LTR gag F AAACATATAGTATGGGCAAGCAG
EXCISION 3'LTR R GCTGCTTATATGCAGCATCTGAGGG
MOGST4 InDel MOGScutF GGGTGGGAAGGAATGGATC
detection
MOGSveroRE CAAGCAAGGAAATCAAGGCTTAG

MOGS1-4 InDel MOGSBF TATACAGGCAGTTGTTTAAGG

detection
MOGS1B ACGTCTACTTCGGCATGAAGAC

2. Taqman ddPCRs

HIV-1 DNA HIV-1/ Ψ F CAGGACTCGGCTTGCTGAAG

HIV-1/ Ψ R GCACCCATCTCTCTCCTTCTAGC

HIV-1/ Ψ Probe FAM TTTTGGCGTACTCACCAGT

Homo sapiens HsTERT F TGGAGCAAGTTGCAAAGCAT

DNA reference
HsTERT R CAGAGCCTTGCACAGAATCC
HsTERT probe HEX CCGGCCTCAGCATGCGCCTG
3. Standard RT-PCRs

SaCas9 SaCas9 F CTGGAACGGCTGAAGAAAGACGG

SaCas9 R CGGTAATGTCCTTGATGTCGTGGTA

gRNAs gRNA LTR F AAAAACCAATCTAACGTTGGG

gRNA Gag F TATACAGGCAGTTGTTTAAGG

pX601gRNAscaffold R CGCCAACAAGTTGACGAGAT

MOGS MOGS F GAAGGGTCTGAGCAGAAGGT

MOGS R AAGCCTCGTGGGAAGAATGA

TatF CATCCAGGAAGTCAGCCTA

31
Tat TatR TGCTCTCCACCTTCTTCTTC

Reference β-actin F CTACAATGAGCTGCGTGTGGC

β-actin R CAGGTCCAGACGCAGGATGGC

4. OFF-target analysis

MOGS-A OFF- MOGSAOTChr1F AAGCCTTCAGGCAGCATCTT

targets
MOGSAOTChr1R ACTGGTCACCGAAGTGTTCA

MOGSAOTChr18F GCCCAACTGGATAAGGACCC

MOGSAOTChr18R GCATGTGGCATCATCAGACC

MOGSAOTChr7F CTGCTTCCCTGCCCACTTCT

MOGSAOTChr7R CGTTCTGTGGCACCATAAGC

MOGSAOTChr2F AGTACGCAGAGAGCTCCCGA

MOGSAOTChr2R CCCAGGTCCATGGAAACCAA

MOGSAOTChr15F CCTCCATCCCTCAGGAGTGC

MOGSAOTChr15R TGCGTGCCCCATTCCTAGTC

MOGS-B OFF- MOGSBOTChr9F CTCGCTCACCGGGGTATCTG

targets
MOGSBOTChr9R CTGGCACAGGTGTCCAGGGT

MOGSBOTChr1F AGGGTTGACCTTGGATGAGC

MOGSBOTChr1R CCCATTAGCCCGGGATCACA

MOGSBOTChr5F TTTGAGAAGCTGGCTGGCGA

MOGSBOTChr5R CTGGCTCCTGGAGGGGTAAC

MOGSBOTChr7F CCTGCTAACCTGCGTTCTGT

MOGSBOTChr7R GGCAGGCTTTGCCTCATTTC

MOGSBOTPRDM6F GTGCAATTACTGCCATGCCT

MOGSBOTPRDM6R CCTTCCTGCCTGTTGCCATT

5. Cloning gRNAs protospacers into pX601 AAV vector

Cloning single MOGS-A gRNA top CACCGAAAAACCAATCTAACGTTGGG

gRNAs into
pX601 vector MOGS-A gRNA bottom AAACCCCAACGTTAGATTGGTTTTTC

MOGS-B gRNA top CACCGTATACAGGCAGTTGTTTAAGG

MOGS-B gRNA bottom AAACCCTTAAACAACTGCCTGTATAC

MOGST2 gRNA top CACCGCTCACACGTGTGCCTGAGCTT

MOGST2 gRNA bottom AAACAAGCTCAGGCACACGTGTGAGCC

32
 MOGST4 gRNA top CACCGATGGGGCCTTAAGGCTCACCA

MOGST4 gRNA bottom AAACTGGTGAGCCTTAAGGCCCCATCC

Multiplexing InFusion/T795/F ATTACGCTTAAGAATTCCTAGAGC

gRNA cassettes
InFusion/T796/R GGAAATAGGCCCTCAGACTAGGGGTTCCTGCGGC
CGCAAA

Cloning U6- Acc65I-U6 F GGCCTCTAGAGGAGGTGTGGCCTGGGC

MOGST4
cassette into pPapi Scaffold-BstBI R TGGCACCGGTTAAGCAGTGGGTTCC
LV vector

6. Cloning LTR promoters

LTR-454/+60 Xba1LTRp(-454)F GGCCTCTAGATGGAAGGGCTAATTTGG

Age1LTRp(+66)R TGGCACCGGTTAAGCAGTGGGTTCC

LTR-120/+60 Xba1LTRp(-120)F GGCCTCTAGATCGAGCTTTCTACAAGG

Age1LTRp(+66)R TGGCACCGGTTAAGCAGTGGGTTCC

LTR-80/+60 Xba1LTRp(-80)F GGCCTCTAGAGGAGGTGTGGCCTGGGC

Age1LTRp(+66)R TGGCACCGGTTAAGCAGTGGGTTCC

7. Synthetic gRNAs for CRISPR-RNP electroporations

SpCas9 gRNAs MOGS1 CGCAGCAGGGCACCACCCCG

MOGS2 GCGCAGCAGGGCACCACCCC

MOGS3 GGCGCAGCAGGGCACCACCC

MOGS4 UCUACCCCCAGGACUGAUGU

SaCas9 GAUGGGGCCUUAAGGCUCACCAGUUUUAGUACU
CUGGAAACAGAAUCUACUAAAACAAGGCAAAAUG
CCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUU
MOGST4 U

GCAGAACUACACACCAGGGCCGUUUUAGUACUCU
GGAAACAGAAUCUACUAAAACAAGGCAAAAUGCC
LTR GUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU

GGAUAGAUGUAAAAGACACCAGUUUUAGUACUCU
GGAAACAGAAUCUACUAAAACAAGGCAAAAUGCC
GagD GUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU

33
Table S3
Mean ± SD (n=3)
Jurkat Jurkat
Group Jurkat wt clone5 clone13 U1 U1 clone1
Live Cells 97.85±0.45 97.57±0.24 97.75±0.2 98.28±0.84 99.02±0.38
Cell Viability Dead Cells 2.15±0.45 2.43±0.24 2.25±0.2 1.72±0.84 0.98±0.38
G0/G1 41.98±0.94 47.53±0.58 43.710.96 48.08±1.21 49.36±2.08
S Phase 22.28±0.41 23.84±0.99 24.12±0.99 31.79±0.80 30.81±0.79
G2/M 32.74±1.36 27.33±0.53 28.42±0.75 14.30±0.78 14.87±2.00
Cell Cycle Sub G0 1.59±0.14 0.79±0.16 2.76±0.07 5.66±0.28 4.80±0.47
Annexin(-) 7-AAD(+) 0.13±0.06 0.10±0.00 0.07±0.06 0.2±0.00 0.23±0.06
Annexin(+) 7-AAD(+) 4.63±0.31 3.03±0.15 5.10±0.17 2.10±0.30 2.30±0.20
Cell Annexin(-) 7-AAD(-) 89.43±0.55 92.93±0.40 86.20±0.46 93.23±0.25 92.57±0.49
Apoptosis Annexin(+) 7-AAD(-) 5.83±0.32 3.97±0.32 8.63±0.60 4.43±0.06 4.87±0.57

34