Artículo CRISPR

Cardiovascular Research (2020) 116, 894–907 REVIEW
doi:10.1093/cvr/cvz250
CRISPR/Cas9 gene-editing strategies in
Downloaded from https://academic.oup.com/cardiovascres/article-abstract/116/5/894/5581353 by Colegio Mayor Universidad del Rosario user on 22 April 2020
cardiovascular cells
1
Eva Vermersch , Charlène Jouve1, and Jean-Sébastien Hulot 1,2
*
1
Paris Cardiovascular Research Center PARCC, Université de Paris, INSERM, 56 Rue Leblanc, 75015 Paris, France; and 2Centre d’Investigations Cliniques CIC1418, AP-HP,
Hôpital Européen Georges Pompidou, 75015 Paris, France
Received 12 December 2018; revised 5 May 2019; editorial decision 1 September 2019; accepted 26 September 2019; online publish-ahead-of-print 18 November 2019
Abstract Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and consid-
ered as a leading public health issue. Therefore, there is a strong need for new disease models to support the de-
velopment of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regu-
larly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of ge-
netically modified cells and organisms with much greater efficiency and precision than before. The simplicity of
CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used
in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel thera-
peutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the
generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts
and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of
CRISPR–Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovas-
cular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We
will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical
limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to de-
velop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA re-
pair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their effi-
ciency and safety.
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* Corresponding author. Tel: þ33 1 56 09 20 17; fax: þ33 1 56 09 20 28, E-mail: jean-sebastien.hulot@aphp.fr
Published on behalf of the European Society of Cardiology. All rights reserved. V
C The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.
Genome editing in cardiology 895
Graphical Abstract
....................................................................................................................................................................................................
Keywords Genome editing • CRISPR/Cas9 • Genetics • Cardiomyopathy • Disease modelling
.. DNA manipulation in yeast3 it has become increasingly feasible during

1. Introduction ..
.. the last decade following the progressive discoveries of ever-faster and
The possibility of manipulating DNA has enabled many advances in car-
.. easier-to-manipulate technologies.
..
diovascular medicine, especially in terms of understanding gene functions .. The first important development came from hybrid proteins, such as
..
and genetic diseases but also of developing new targeted therapies.1 In .. zinc finger nucleases (ZFNs) and transcription activator-like effector
the future, there is some hope that genome editing tools could poten- .. nucleases (TALENs), which are specifically designed and engineered
..
tially be developed into therapies to cure certain genetic forms of cardio- .. through recombination and have a nuclease module associated with rec-
vascular diseases.2 .. ognition sites to bind a targeted DNA sequence. After binding to the
..
In general, genome editing refers to the specific modification of DNA .. recognition site, these proteins create a DSB of the DNA. The ZFNs are
following the action of nucleases. These nucleases cut a DNA sequence .. artificial nucleases created by the fusion of zinc finger motifs with a Fok I
..
at a specific targeted point thus creating a DNA damage [typically a dou- .. cleavage domain,4 responsible for the DNA cleavage as long as two Fok I
ble strand break (DSB)] which in turn triggers a natural DNA repair .. domains dimerize.5 Transcription activator-like effectors (TALEs) are
..
mechanism. As detailed later in this review, the process of natural DNA .. effectors proteins that have the ability to bind promoter regions in the
repair can be repurposed in order to make precise genome manipula- .. nucleus of the host, and activate gene expression.6 TALE proteins pro-
..
tions or DNA edits. While genome editing began in the late 1970s with . vide a programmable module for DNA binding and, fused with a Fok I
896 E. Vermersch et al.
..
module, become an interesting genome editing tool. The hybrid protein .. DNA. The mature crRNA, along with a trans-activating crRNA
forms a TALE/nuclease (abbreviated as TALEN). TALENs are, like .. (tracrRNA),22 will be used as a small guide18 for the ribonuclease Cas9
..
ZFNs, capable of creating DSBs in specific locations in the genome when .. which will introduce DSB 3-nt upstream of a typical sequence referred
they dimerize.7 Of importance, TALEN endonucleases have reached .. as the protospacer adjacent motif (PAM). The PAM sequence is a short
..
clinical experimentation8 and are particularly used for the generation of .. nucleotide motif adjacent to the target DNA sequence23 and mandatory
universal allogeneic CAR T-cells product for B-cell lymphoma.9 .. for the Cas9 nuclease activity. Importantly, the tracrRNA–crRNA com-
..
Most of the recent expansion of genome editing is however associ- .. plex could be substituted by a single guide RNA (sgRNA)15,20 and, thus,
ated to CRISPR/Cas9 technology.10 Indeed, CRISPR/Cas9 represents a .. the nuclease activity will be achieved with only two elements: the Cas9
..
potent and simpler technique to perform genome editing in vitro and .. protein [typically Streptococcus pyogenes (spCas9) as one of the first de-
in vivo. CRISPR/Cas9 has thus been widely adopted by the scientific com- .. scribed and cloned15] and a specific guide RNA (Figure 1A).
..
munity. However, as for other genome editing tools, the success of ge- .. Therefore, the natural CRISPR/Cas9 system has been modified to
nome editing using CRISPR/Cas9 does not only result from its nuclease .. form an innovative genome editing tool that is based on the combination
..
action but also from the subsequent activation of specific mechanisms to .. of Cas9 and guide RNAs (or sgRNA) which are specifically designed to
repair the damaged DNA. The efficiency of these repair mechanisms can .. target a given DNA site. The design of sgRNAs can be performed using
..
be variable, notably in differentiated cells. For instance, homology- .. different bioinformatic tools which predict efficiency and specificity (or
directed repair (HDR, an error-free DNA repair mechanism) has been
.. off-target effects).24,25 Last, multiplex genome editing can be achieved by
..
extensively used for precise gene editing but is restricted to the S and .. administrating only one Cas9 protein injection with multiple designed
G2 phases of the cell cycle. This approach might not be suitable for edit-
.. sgRNAs. Thus, it is possible to simultaneously edit several DNA
..
ing and repairing the genome of non-dividing cells such as adult cardio- .. sequences.20,26
myocytes, which could however be edited with other DNA repair
.. Different CRISPR–Cas nucleases have been reported to be efficient in
..
pathways. In addition, most strategies (such as HDR) require a donor .. mammalian cells and can represent alternatives to the original spCas9 to
..
template to perform precise gene editing, thus adding a level of complex- .. perform genome editing as they notably recognize different PAM
ity on the design and delivery of the material to the targeted cells. To cir- .. sequences27 (Table 1). Thus, the use of additional Cas9 nucleases widens
..
cumvent these limitations, new donor-independent tools, such as base .. the number of sites amenable to genome editing in mammalian cells. In
editors, have been proposed, thus expanding the options to perform ge- .. addition, other types of natural and engineered CRISPR systems con-
..
nome editing. .. tinue to be described. The deactivation of one of the two catalytic sites
Here, we review the recent development of CRISPR/Cas9 genome .. creates a new version of Cas9 called ‘nickase’. As compared to the origi-
..
editing, the alternative tools, the available strategies to conduct genome .. nal Cas9, DSB will be achieved by designing two different sgRNAs: one
editing in cardiovascular cells, and the current and future applications in .. to target and break the forward strand and the second one for the re-
..
cardiovascular biology. .. verse strand. The utilization of nickase allows breaks with sticky end and
.. a more specific targeting, limiting off-target effects.28 The Class 2
..
.. CRISPR/Cas12a (formerly known as Cpf1) is another single RNA-guided
.. endonuclease that requires only a single short 40 nt crRNA29 and recog-
2. RNA-guided endonucleases and ..
.. nizes a T-rich PAM sequence29 (whereas Cas9 needs a G-rich sequence).
variants .. Although Cas12a enzymes have shown useful for genome editing,30 their
..
.. efficiency was lower than Cas9 nucleases but new engineered Cas12a
2.1 CRISPR/Cas9 and others DNA .. variants with enhanced activities have been recently described.31
..
nucleases ..
CRISPR/Cas9 refers to an adaptive immune system initially discovered in
..
..
bacteria. CRISPR stems for ‘clustered regularly interspaced short palin- .. 2.2 Consequences of DSBs & mechanisms
dromic repeats’ that were reported in different bacterial species11,12 and
.. of DNA repair
..
consist of short arrays of repeated sequences separated by specific .. DNA DSBs can be repaired by endogenous cellular repair pathways:
..
spacer sequences. As such, CRISPR does not refer to a genome editing .. non-homologous end joining (NHEJ) and HDR. NHEJ is the dominant
technology but to the storage of snippets of invading viruses’ DNAs into .. pathway but is generally recognized as an error-prone pathway. NHEJ
..
the bacterial genome.13 CRISPR were later shown as key elements in .. occurs independently of a donor template and results in various length
bacterial immune response against viruses and plasmids14,15 in associa- .. small insertions or deletions (indel) at the site of DNA repair32
..
tion with CRISPR-associated proteins16 (Cas). Cas proteins family is a .. (Figure 1A).
large and heterogeneous family of proteins, including nucleases, heli- .. HDR is another pathway where the DSB site is rebuilt using a DNA
..
cases, and polymerases.17 There are numerous CRISPR/Cas systems, .. template which can either be the homologous chromosomal DNA (i.e.
classified in classes according to the number of effectors, types, and sub- .. sister chromatids) or an exogenous template DNA strand. HDR
..
types.17 Cas9 is a Class 2 system which is composed of a single-subunit .. involves a specific machinery to remodel the chromatin and recruit
effector, which will be encoded by a single gene thus simplifying its use. .. DNA repair enzymes.33 Then, homologous recombination repair can
..
The mechanism by which the CRISPR/Cas9 dual component bacterial .. naturally occur through different mechanisms among which the
system works has been reviewed elsewhere18 and will not be developed .. synthesis-dependent strand annealing (SDSA) and the double strand
..
here. However, the relevance of this system to biology and medicine .. break repair (DSBR) pathways are the most widely recognized.34 Briefly,
stems from the observation made in 2013 that CRISPR/Cas9 is also ac- .. these two mechanisms share common initial steps where a resection of
..
tive in mammalian cells.19–21 In bacteria, CRISPR sequences are .. the 50 ends of the DNA break will first be performed, then followed by
expressed into CRISPR RNA precursors (pre-crRNA) that are then
.. invasion of a DNA strand similar to one of the 30 overhangs. Once strand
..
cleaved to become a CRISPR RNA (crRNA), base-paired to the invader . invasion has occurred, a displacement loop is formed and will be
Figure 1 Different applications of genome and epigenome editing with Cas9 and Cas9 variants. (A) Wild-type Cas9 nuclease combines with a guide RNA
(gRNA) to create a double strand break in the targeted DNA sequence. This double strand break can be repaired by endogenous cellular repair pathways:
non-homologous end joining (NHEJ) and homology-directed repair (HDR). (B) dCas9 (for dead Cas9) is created after deactivation of the catalytic domain
via targeted mutagenesis. The catalytically inactive dCas9 becomes an RNA programmable DNA binding domain and can block transcription initiation or
elongation by targeting a promotor or the coding sequence of a gene. (C) dCas9 fused to a transcription repressor (such as KRAB effector domain) to meth-
ylate histones and block transcription. (D) dCas9 fused to a transcriptional activator domain. Different activators have been proposed such as VP64 [four vi-
rion protein 16 (VP16) linked with Gly-Ser linkers], VPR (VP64-p65-Rta) or SunTag (a repeating polypeptide array with multiple copies of antibodies
attached to transcription activators).
Table 1 Characteristics of main Cas enzymes
Species of Cas9 Size of the coding sequence (kb) Recognized PAM sequence
..............................................................................................................................................................................................................................
Streptococcus pyogenes (SpCas9) 4.10 30 NGG
Staphylococcus aureus (SaCas9) 3.16 30 NNGRRT or NNGRR(N)
Acidaminococcus sp (AsCpf1) and 3.92 & 3.68, 50 TTTV
Lachnospiraceae bacterium (LbCpf1) respectively
Campylobacter jejuni Cas9 2.95 30 NNNNRYAC
Neisseria meningitidis CasS9 3.24 30 NNNNGATT
Streptococcus thermophilus Cas9 7.60 30 NNAGAAW
extended by DNA synthesis with the involvement of different sets of re- .. crossover and non-crossover products. However, in mitotic cells, ho-
..
pair proteins for NHEJ and HDR. The second end extension and ligation .. mologous recombination is mainly achieved via SDSA which is a model
result in DNA repair through the DSBR pathway which either ends with
.. of non-crossover recombination.
.
..
As HDR needs a homologous duplex DNA template to create an ac- .. 2.3.2 Gene activation, repression, and epigenome editing
curate repair, HDR is considered as error-free. HDR is the preferred .. using CRISPR–Cas9 system
..
mechanism to perform precise gene editing and DNA repair but HDR is .. The dCas9 could also be fused with other modules allowing different
limited by several factors.34,35 First, HDR is variably efficient and typically .. functions. For instance, dCas9 has been fused with transcriptional re-
..
occurs at lower efficiency rates as compared to NHEJ in somatic cells. In .. pressor or activator (Figure 1C and D). In the repression system, dCas9 is
mammalian cells, HDR events are thus relatively rare. Secondly, the use .. fused with a transcriptional repressor such as KRAB or KRAB–MeCP2
..
of HDR is largely determined by the phase of cell cycle.35 Homologous .. fusion protein (a more potent repressor) to methylate histones and
recombination repairs DNA before the cell enters mitosis (M phase) .. block transcription thus creating a reversible knockdown. This system,
..
and is thus restricted to the S and G2 phases36 of the cell cycle. .. referred to as CRISPR interference (CRISPRi—by analogy with RNAi),
Reciprocally, NHEJ is predominant in the G1 phase of the cell cycle. It is .. offers the opportunity to down-regulate multiple genes at the same time
..
fast, efficient, flexible, and the predominant form of DSB repair but is er- .. by using multiple sgRNAs that will convey CRISPRi to multiple loci.38 In
ror prone. In addition to the cell cycle, different other factors such as the .. the same way, when dCas9 is fused with an activator domain such as
..
type of cells and the localization of the DSB37 regulate the choice be- .. VP64 [four virion protein 16 (VP16) linked with Gly-Ser linkers], the sys-
tween NHEJ and HDR.33 Thirdly, the desired genome editing outcome ..
.. tem can activate gene expression. This system is referred to as CRISPRa
will also influence the preferred mechanism. Indeed, NHEJ is widely used .. (activation).41–43 In contrast to the genome editing and base editing
for knock-out strategies in stem cells and as well as in somatic cells and
..
.. tools, these later dCas9 variants do not perform irreversible changes to
some therapeutic strategies have been developed using NHEJ as later .. the DNA but rather offer reversible modulation of gene expression.
explained.
..
.. These systems can also be used for large-scale gene expression studies
.. by combining CRISPRi or CRISPRa with gRNA screening libraries.
..
..
..
2.3 Variants to CRIPSR/Cas9: base editors ..
..
..
3. Strategies to perform genome
and others
2.3.1 Single nucleotide editing
..
.. editing in heart cells
Further developments in genome editing strategies come from the tar- ..
.. 3.1 Experimental considerations
geted modifications of the original CRISPR/Cas9 system, notably through ..
the deactivation of the catalytic domains (forming a so-called dead Cas9 .. 3.1.1 Type of cells
.. During these last years, CRISPR/Cas9 gene editing has been largely used
or dCas9)38 (Figure 1B). The dCas9 variant keeps its ability to bind to a ..
given DNA target sequence in combination with a sgRNA but does not .. in very different cellular sources including germ cells and developing
.. zygotes, pluripotent stem cells that are redifferentiated into cardiovascu-
cleave the DNA anymore. In addition, new functional modules can be ..
added to the dCas9. Using dCas9, a new approach called ‘base editing’ .. lar cells of interest44,45 and terminally differentiated cells. There are how-
.. ever technical and ethical (discussed later) issues that depend on the
has recently been reported as allowing direct replacement of a single ..
base pair in a genome without the need for DNA cleavage. Base editing .. type of cells considered for genome editing. First, as already explained,
.. the efficiency of the different DNA repair mechanisms varies substan-
was first reported using a dCas9 fused with a cytidine deaminase enzyme ..
that enables C–G to T–A conversion39 (cytosine base editors). Further
.. tially by cell type and cell state. NHEJ can effectively generate indel muta-
.. tions in most cell types and has thus been performed in a variety of cell
engineering experiments lead to the more recent development of ade- ..
nine base editors which enable an A–T to G–C transition.40
.. types including dividing and post-mitotic cells. In contrast, HDR-
.. mediated editing is generally thought to be more efficient in proliferating
The development of these new base editors represents a major mile- ..
stone in genome editing for three main reasons. First, base editing is inde-
.. cells. As a consequence, HDR is uncommon in somatic cells, thus limiting
.. precise genome editing in differentiated cells with limited proliferation,
pendent of DSB and therefore escapes limitations of HDR and ..
promiscuity of NHEJ as discussed previously. Base editors can therefore
.. such as human cardiac myocytes.46 Secondly, the delivery of program-
.. mable nucleases largely depends on the target cells. In general, strategies
represent a suitable approach to perform genome editing in dividing as ..
.. involving ex vivo genome editing of cultured cells are easier to control
well as non-dividing cells. Secondly, base editing is donor-independent as ..
nucleotide change results from the direct enzymatic action of the engi- .. and achieve higher editing rates. Edited cells can then be kept into culture
.. or retransplanted into an organism. Germ cells and other stem cells are
neered dCas9 variant, thus representing a technical advantage over ..
donor-dependent strategies. Thirdly, target mutations C–G to T–A ac- .. particularly appropriate for such approaches. In contrast, in vivo genome
.. editing involves direct delivery of the genome editing tools to the af-
count for half of the known pathogenic point mutations in humans.40 ..
Base-editing approaches can currently be used to target T > C, A > G, .. fected somatic cells in their native tissues which can be extremely chal-
.. lenging and dependent on the delivery vector and mode of
C > T and G > A substitution mutations, representing a significant num- ..
ber of the pathogenic variants in human genome. In addition, base editing .. administration. Thirdly, DNA modifications in germ cells are permanent
.. and can be passed to future generations, thus creating considerable de-
can also be used to engineer a premature stop codon thus preventing ..
the expression of the mutated exon (Figure 2). Moreover, base editing .. bate about the ethics of germline genome editing, especially in humans.47
.. From an ethical viewpoint, somatic genome editing is thus more widely
has limited off-target effects as compared to the conventional CRIPSR– ..
Cas9 systems. Despite the higher fidelity of base editing, expression of .. accepted as the DNA modifications will treat the affected individual
.. without transmission to future descendants.
the deaminases, such as cytosine deaminase, could induce spurious off- ..
target editing. Other shortcomings of the base editors include sequence ..
..
preference, as efficiency of some base editors varies according to the .. 3.1.2 Type of mutations
composition of nucleotide downstream to the targeted site, inaccessibil-
.. Genome editing approaches are designed according to the nature of the
..
ity of certain sites, and occasional unexpected mutagenesis. . target mutations and the desired outcome. Figure 2 summarized the
Figure 2 Genome editing strategies according to the target mutation. The main approaches (in bold letters) are listed in the left, the corresponding muta-
tions in the middle and a schematic representation of the genome editing outcome is reported on the right. For the example, exons with a pathogenic muta-
tion are in red, the black star indicating the mutation. Non-mutated exons are in white and corrected exons provided as a template are in green. Green
arrows indicate the manipulation sites. GOF, gain-of-function HDR, homology-directed repair; NHEJ, non-homologous end joining.
main approaches developed thus far. A first approach is based on gene

.. performed by introducing a copy of the wild-type gene at the mutated
..
disruption where a gene of interest (GOI) is silenced through the forma- .. locus or in a safe harbour locus.
tion of indels using NHEJ. Genome editing tools can be designed to in-
..
..
duce DSB near the starting codon and formation of indels often results ..
..
in frameshift mutations that create premature stop codon ensuring gene .. 3.1.3 Delivery of the components
knock-out.48 Of note, the newer base editors can also be used to di- .. Delivery of the genome editing package to the target cell is a key chal-
..
rectly introduce a premature stop codon and disrupt the GOI expres- .. lenge, notably for in vivo genome editing. The different delivery options
sion. NHEJ can also be used to perform deletion of a DNA sequence via .. for clinical translation have been recently reviewed elsewhere53 and the
..
the creation of two DSBs on both sides of a pathogenic mutation.49–52 .. most appropriate techniques for genome editing will be briefly described
This approach will require multiplexed targeting that can be achieved .. in this section.
..
with two different guide RNAs driving the Cas9 to two specific flanking .. In ex vivo editing therapy, cells can be manipulated with a wide range of
sequences. Exon skipping of the Duchenne muscular dystrophy (DMD) .. delivery techniques, such as electroporation, micro-injection, transfec-
..
pathogenic exon 51 was however recently achieved using a single guide .. tion agents, cell-penetrating peptides, and viral vectors.54 The CRISPR
RNA that created reframing mutations, thus showing that the approach .. components can notably be found into different formats including plas-
..
can even be optimized to achieve efficient gene deletion.50 Point muta- .. mids, mRNAs, and ribonucleoprotein complex (RNP). CRISPR/Cas9
tions can be corrected by two different approaches. Most of the studies .. RNP is a DNA-free format that was recently shown to provide higher
..
performed so far have used HDR which requires a donor template (i.e. .. editing efficiency and fewer potential off-target effects in a variety of cell
the wild-type sequence in the current example). Newer base editors .. types.55 The RNP approach indeed allows the genome editing compo-
..
have been proposed to perform precise nucleotide substitution without .. nents to be transiently expressed and then quickly cleared from the cells
the need for a donor template. Lastly, gene addition via HDR can be
.. by degradation.
..
The transient expression of the genome editing components is prefer- .. zygotes micro-injections. This is in line with the overall observation that
able to avoid unintended editing outcomes,56,57 however representing a .. genome editing events are rare, which can however be compensated by
..
key challenge for in vivo genome editing. Viral vectors such as recombi- .. an increase in the number of treated cells which is affordable when ma-
nant adeno-associated viral (rAAV) serotypes offer the advantage of a .. nipulating dividing cells such as stem cells.
..
natural tropism for specific cell types (including muscular cells) but in- .. Beyond animal models, CRISPR/Cas9 can also be developed as a ther-
duce long-term transgene expression. AAV8 and AAV9 have been nota- .. apeutic tool for inherited cardiac diseases by performing germline ge-
..
bly used to perform DMD genome editing in small and large animals, .. nome editing. CRISPR/Cas9 genome editing was performed to correct a
either through intra-muscular delivery or systemic delivery.50–52 Non-vi- .. dystrophin gene mutation (e.g. in exon 23) in the germline of mdx mice, a
..
ral gene delivery techniques could also prove useful for in vivo genome .. classical model of DMD.49 DNA sequencing revealed a wide range of
editing. Modified RNAs trigger high-level but short-term transgene ex- .. mosaicism (i.e. 2–100% correction of the DMD gene) through either
..
pression and can be delivered to the heart.58 Cas9-sgRNA ribonucleo- .. NHEJ or HDR mechanisms. However, even a low rate of correction was
proteins can also be directly used for in vivo genome editing,59,60 as .. sufficient for complete phenotypic rescue,49 which can be explained by a
..
shown in mouse embryos and in the liver of tyrosinaemic FRG mice.59 In .. survival advantage and subsequent expansion of edited cells within the
addition, using non-viral delivery technique can solve the packaging limit .. muscle satellite cell population.
..
(i.e. 4.8 kb) of AAVs which are generally too small to package the original .. Similarly, a correction of an MYBPC3 mutation causing hypertrophic
S. pyogenes Cas9 (Table 1).
.. cardiomyopathy (HCM) was recently achieved in human germ cells using
..
Lastly, transgenic animals stably expressing Cas9 have been recently .. CRISPR/Cas9. In this study,71 a short-lived recombinant Cas9 protein/
developed and simply require guide RNAs to be cloned and delivered to
.. RNA complex was micro-injected into M-phase oocytes at the same
..
the target organ to study gene function.61,62 In mice with cardiomyocyte .. time as the sperm from a male donor who was heterozygous for
specific expression of Cas9, long-term expression of Cas9 does not af-
.. MYBPC3 mutation. This strategy resulted in significant increases in HDR
..
fect cardiac function or gene expression and these mice were used for .. efficiency as well as a large reduction in mosaicism as gene correction oc-
.. 71
subsequent and efficient gene editing of cardiac genes including Myh6, .. curred before the fertilized eggs started dividing. In another example
Sav1, and Tbx20.61 Similarly, Wang et al. have developed Cre-dependent .. of human germline editing, Zeng et al. used base editors to perform al- 72
..
Cas9-expressing pigs.63 .. lele correction of a point mutation (i.e. FBN1T7498C) associated with
.. Marfan syndrome. Mature oocytes were fertilized with the sperm from a
..
.. male donor who was heterozygous for FBN1 mutation and these
3.2 Examples in germline cells & stem cells .. zygotes were micro-injected with the mRNA of BE3 and the correc-
..
3.2.1 Embryonic stem cells .. tional sgRNA. The BE3 system was thus able to introduces a C > T con-
The progressive development of genome editing tools has clearly facili- .. version with a high efficiency rate, thereby expanding the applications of
..
tated the generation of knock-in and knock-out animals, particularly in .. base editors.72
rodents.64 The rise of CRISPR/Cas9 has particularly revolutionized the ..
..
generation of transgenic animals because of its higher efficiency to per- .. 3.2.2 Induced pluripotent stem cells
form site-specific DNA manipulation. In these experiments, the common .. One of the main reasons for the large adoption of genome engineering
..
process is to micro-inject the genome editing material (e.g. Cas9, .. in human induced pluripotent stem cells (hiPSCs)73–75 comes from the
sgRNAs, and single-stranded oligo donor as template) in a single step
.. need to deal with the influence of inter-line variability on the observed
..
into the animal zygotes, then re-implant these genome-edited zygotes.64 .. cellular phenotypes. There are indeed multiple genetic and epigenetic
The genome-edited pups are then selected through genotyping as cor-
.. differences between cell lines and the reprogramming itself is responsible
..
rected animals will show mosaicism with different levels of correction .. for epigenetic variability.76 To overcome this issue, appropriate control
between cells with the presence of corrected and non-corrected nuclei
.. cell lines are generated that have the same genetic background as the
..
within an organ.65 This mosaicism is attributed to the inability of CRISPR/ .. cell line carrying the pathogenic variant (isogenic cell lines). Isogenic
Cas9 to correct all mutant genes after cell division occurred.
.. hiPSCs are considered as the gold standard in disease modelling studies
..
In addition, CRISPR/Cas9 has been shown to be applicable to a wide .. using hiPSCs. While the initial concept was to specifically correct a given
..
variety of animal species, thus expanding the realm of animal models in- .. mutation in patient-specific hiPSCs, the emergence of CRISPR/Cas9 has
cluding small animals such as zebrafish,66 rats,67 or large animal such as .. also offered a unique opportunity to introduce a specific mutation.77–79
..
pigs.68 This approach will have important consequences as human car- .. in a normal hiPSC colony, thus speeding up the process for functional
diac disorders can be incompletely recapitulated in some animal models. .. screenings in a patient-independent model.
..
For instance, the study of DMD has been for a long-time limited to three .. Various studies have been done in the cardiovascular field with
major animal models (mice, pigs, and dogs) but is now complemented by .. patient-specific hiPSCs and are reviewed elsewhere.45,75,80 Single genetic
..
the development of a new rabbit model via CRISPR/Cas9 thus offering a .. mutations responsible for cardiomyopathies, such as dilated cardiomy-
new alternative for preclinical studies.69 .. opathy,81 HCM,78,82 Barth syndrome,83 long-QT syndrome,84,85 and
..
CRISPR/Cas9 genome editing can also be performed using zygotes .. DMD,86 have been corrected by genome editing in patient-specific iPSC-
from already generated transgenic animals. To further explore the role .. derived cardiomyocytes. A recent publication further highlights the use
..
of phospholamban (PLN) in the development of heart failure, Kaneko .. of CRISPR/Cas9 to investigate variants of uncertain significance (VUS)
et al.70 performed germline genome editing to achieve PLN ablation in .. likely associated with a cardiac channelopathy.87 In this study, two sour-
..
calsequestrin overexpressing transgenic mice (CSQ-tg mice). The calse- .. ces of iPSCs were used: one from a control subject and another from a
questrin model was selected as mice develop severe heart failure and .. patient carrying a VUS in KCNH2. CRISPR/Cas9 genome editing was
..
the study showed that PLN ablation improved mice survival and cardiac .. used to introduce the homozygous variant in the control cell line and to
function.70 Interestingly, the yield was low (i.e. about 1%) with the gener-
.. correct the mutation in the patient-specific cell line, thus providing two
..
ation of 40 PLN homozygous KO/CSQ-tg mice over a total of 4051 . independent pairs of isogenic control and mutant cells to compare
Table 2 List of current strategies to perform in vivo genome editing for the cardiac muscle
Genome editing in cardiology
Pathology (targeted Animal Editing strategy Packaging Delivery: Maximal efficiency Maximal Off-target Immune response References
gene) model route and age (% of edited CM) follow-up events
...........................................................................................................................................................................................................................................................................................................
Duchenne muscular Mdx mice Deletion of mutated Dual AAV9: IM at P12 – 12 weeks None (top 10 pre- – Long et al.52
dystrophy (Dmd) exon 23 via NHEJ SpCas9 RO at P18 9.6 ± 3.9% dicted sites)
2 SgRNAs IP at P1 3.2 ± 2.4%
Dual AAV9: IM in adults – 3 weeks None (top 8 pre- – Tabebordbar
SaCas9 IP at P3 5% dicted sites) et al.89
2 SgRNAs IV in adults 10–20%
Dual AAV8: IM, 8w-old – 1 year 1% at one of the Humoral response in Nelson
SaCas9 IP at P2 6% predicted site, adult mice, not in et al.90,92
2 sgRNAs IV, 8w-old 30% undetectable at neonates
other sites
Mdx/Utrþ/ mice Deletion of mutated Single AAVrh74: RO/IP at P3 39.0 ± 0.4% 10 weeks None Not observed Refaey et al.91
exon 23 via NHEJ Sacas9/2 sgRNAs IV, 16w-old 40%
DEx50 mice Skipping of exon 51 Dual AAV9: IM at P12 Yes, 40% 8 weeks None (top 6 pre- Not observed Amoasii et al.50
via NHEJ SpCas9 IP at P4 20% dicted sites)
1 SgRNA
DE50-MD dogs Skipping of exon 51 Dual AAV9: IM, 1-mo old – 8 weeks None (top 4 pre- Immunosuppression Amoasii et al.51
via NHEJ SpCas9 IV, 1-mo old 92% (with high dicted sites) protocol adminis-
1 SgRNA dose AAV9) tered to dogs
PRKAG2-related Wolff– þ/H530R mice Disruption of mutated Dual AAV9: IVe at P4 6.5% 8 weeks None (top 10 pre- – Xie et al.93
Parkinson–White syn- allele via NHEJ SpCas9 IV at P42 2.6% dicted sites)
drome (Prkag2) 2 SgRNAs
Cathecolaminergic poly- R176Q/þ mice Disruption of mutated Single AAV9: SC at P10 20% 6 weeks None – Pan et al.94
morphic ventricular allele via NHEJ SaCas9/1 sgRNA
tachycardia (Ryr2)
IM, intra-muscular; IP, intra-peritoneal; IV, systemic delivery via tail vein injection; IVe, intra-ventricular; NHEJ, non-homologous end joining; P, post-natal day; RO, retro-orbital; SaCas9, Staphylococcus aureus Cas9; SC, subcutaneous; SpCas9,
Streptococcus pyogenes Cas9.
901
..
phenotypic reproducibility. The study of each modification on iPSC- .. ryanodine receptor type 2 (RYR2) causative for CPVT was similarly cor-
derived cardiomyocytes showed that the VUS was likely responsible of a .. rected via a single administration of AAV9-Cas9/SgRNA in neonatal ge-
..
significant prolongation of the action potential duration, and thus, shed- .. netically modified mice (10 days after birth). Gene correction was highly
ding light on the functional impact of the variant. .. specific of the RYR2 mutation without detectable off-target mutations
..
.. and was associated with a strong decrease in spontaneous arrhythmias.
.. Noteworthy, the Staphylococcus aureus Cas9 (saCas9) was used in the
3.3 Examples in somatic cells ..
.. study, as representing a smaller Cas9 orthologue and allowing SaCas9
3.3.1 Cardiac muscle .. and sgRNA to be packaged in a single AAV vector.94
..
Achieving effective in vivo genome editing of cardiac muscle seems ex- ..
tremely challenging because of the difficulties to deliver the genome edit- .. 3.3.2 Other organs
..
ing material to the cardiac tissue and to activate HDR in non-dividing .. Other examples of in vivo genome editing for cardiovascular diseases in-
cells such as cardiomyocytes.46 Table 2 summarizes the current attempts .. clude the edition of genes involved in lipid metabolism. In contrast to
..
for performing in vivo genome editing in the cardiac muscle. The use of .. previous studies directly targeting the cardiac tissue or cardiomyocytes,
CRISPR/Cas9 as a therapy for cardiovascular disorders is only progres-
.. genome editing is here achieved by targeting the liver, a tissue that is less
..
sively emerging, with a primary focus on debilitating forms of diseases .. challenging for gene transfer via viral or non-viral vectors.95 In addition,
such as DMD. DMD is caused by the absence of dystrophin which can
.. these approaches can be developed to cure individuals with monogenic
..
be due to genetic mutations that change the reading frame and create a .. disorders (e.g. familial hypercholesterolaemia) but could also be pro-
premature stop codon.88 However, as a very large gene, many regions of
.. posed to a larger population of patients with high atherosclerotic risk for
..
the protein are dispensable. Consequently, by deleting the mutated se- .. a preventive purpose.
..
quence it is possible to partially restore the dystrophin function.49,52,88 .. The most advanced example relates to the disruption of the propro-
Initial studies52,89,90 showed that the systemic delivery of CRISPR/Cas9 .. tein convertase subtilisin/kexin type 9 (PCSK9), as loss-of-function muta-
..
system packaged into AAV8 or AAV9 to adult or neonatal mdx mice par- .. tions in PCSK9 are associated with reduced risk for coronary heart
tially restored dystrophin expression and improved muscular function. .. disease and pharmacological inhibition of PCSK9 affords clinical benefits
..
Significant recovery of dystrophin expression in the cardiac muscle was .. in high-risk CAD patients.96 PCSK9 gene disruption via NHEJ was re-
also observed in the weeks following AAV-Cas9 administration,90 which .. cently achieved in adult mice using either adenovirus or AAV vectors
..
was associated with a significant improvement of cardiac myofiber archi- .. and showed reduced plasma PCSK9 and total cholesterol levels.97,98
tecture, cardiac fibrosis, and papillary muscle contractility.91 In a recent .. PCSK9 levels were reduced by >90% and total plasma cholesterol by
..
report, it was further shown that protein restoration is sustained in the .. 40% in edited mice. The gene editing efficiency was globally higher than
treated animals for 1 year.92 In these studies, genome editing was .. observed in the cardiac tissue, even when administered in older animals.
..
achieved through NHEJ to delete the mutant exon 23, upon delivery of .. This result is probably linked to a higher proliferative and regenerative
the AAV-Cas9 system in neonates, which led to detection of dystrophin .. state of the liver tissue as compared to cardiac muscle. In line with this
..
expression in up to 40% of cardiomyocytes.91 Similar results were ob- .. assumption, hepatic disruption of the LDL receptor with AAV2/8-
served in a different study which utilized a CRISPR/Cas9 strategy to skip .. CRISPR was achieved with a high efficiency in adult (e.g. 6–9 weeks old)
..
another DMD mutation (i.e. pathogenic exon 51) using a single RNA .. mice.99 Similar results were observed for genome editing of transthyretin
that created reframing mutations.50 In a recent study, Amoasii et al.51
.. (Ttr) gene in the liver of 6–10-week-old mice via a single administration
..
showed increased expression of cardiac and skeletal muscle dystrophin .. of CRISPR/Cas9 encapsulated in lipid nanoparticles.100
in a dog model of DMD after treatment with intravenous AAV9 contain-
.. Another promising technique that could overcome some of these lim-
..
ing Cas9 and gRNA. Noteworthy, these studies used large doses of .. itations is in vivo base editing. A base editor (BE3) was recently used to in-
AAV9 (i.e. from 1 1013 to 2 1014 vg/kg) suggesting that further op-
.. troduce site-specific PCSK9 non-sense variants.98 Because of its large size
..
timization of the method should be required before translation to .. (5.1 Kb), BE3 was packaged in an adenovirus which was administered
humans. In addition, this CRISPR/Cas9-mediated gene editing was re-
.. via retro-orbital injection to 8-week-old mice. On average, base editing
..
sponsible for a genetic mosaicism with a resulting mixture of both edited .. of Pcsk9 resulted in 56% reduction of plasma PCSK9 protein levels and
..
and non-edited cells. Interestingly, the degree of muscle phenotype res- .. 28% reduction of plasma cholesterol levels, a slightly lower extent than
cue in mosaic mice exceeded the efficiency of gene repair,89–91 suggest- .. the one observed with Cas9 nucleases.97 Similarly, reduced blood lipid
..
ing that even an imperfectly efficient genome editing could provide .. levels were achieved with in vivo base editing of ANGPTL3 in 5-week-old
important clinical benefits. .. wild-type or hyperlipidaemic Ldlr-knock out mice.101 In these studies,
..
Other studies aimed to correct disease-causing allele in cardiomyo- .. base editing activity was observed in the liver but not in other organs
cytes using CRISPR/Cas9 system with AAV9 vectors either in neonatal .. such as lung or heart, a result that could be either due to the delivery
..
or in young mice. The Protein Kinase AMP-activated Non-Catalytic .. mode (use of adenoviruses, which poorly transduce cardiac myocytes
Subunit Gamma 2 (PRKAG2) cardiac syndrome is characterized by gly- .. in vivo) or to a lower activity of base editors in those tissues. In a recent
..
cogen accumulation in the cardiac tissue, leading to left ventricular hy- .. proof-of-concept study using BE3 for in utero (i.e. embryonic Day 16)
pertrophy and ventricular pre-excitation. Post-natal correction of a .. base editing of murine Pcsk9,102 a robust and persistent editing was ob-
..
PRKAG2 mutation (e.g. p.His530Arg) was achieved in transgenic and .. served in the liver without evidence of editing in other organs. The study
knock-in mice via a single administration of Cas9/sgRNA in an AAV9 vec- .. was further extended to another metabolic gene (i.e. Hpd) with similar
..
tor either at Day 4 (i.e. when cardiomyocyte proliferation is still occur- .. results. However, BE3 expression was reported in the heart which sug-
ring) or Day 42 (i.e. when cardiomyocytes have ceased to proliferate) .. gests that base editing of cardiomyocytes could be achievable before
..
after birth. In vivo genome editing resulted in significant reduction in left .. birth. A recent direct comparison of BE3 with Cas9 nuclease to perform
ventricular wall thickness and normalization of ECG abnormalities.93 In
.. PCSK9 editing in vivo showed that base editing resulted in a more pro-
..
another study,94 a missense mutation (e.g. p.Arg176Gln) in the . nounced reduction in plasma PCSK9 levels relative to high frequency of
..
editing at the genetic level.103 Overall, this later study showed that base .. composed of less than the regular 20 nucleotides, could increase
editing was as efficient as Cas9-mediated genome editing but was more .. CRISPR/Cas9 specificity by decreasing the off-target frequency.113
..
precise by introducing site-specific point mutations without relying on .. In addition to sgRNA design tools, the occurrence of off-target events
the error-prone NHEJ pathway. .. can also be limited by the use of engineered nucleases. One of the popu-
..
Further studies are now required to optimize in vivo base editing into a .. lar option is the use of paired nickases,28 as earlier presented.
variety of organs, including the cardiac muscle. .. Alternatively, the use of a modified Cas9, such as eSpCas9114 (enhanced
..
.. S. pyogenes Cas9) or Cas9-HF1115 (Cas9 high fidelity), could also reduce
.. off-targets. These engineered Cas9 were generated via punctual muta-
..
4. Future perspectives and concerns .. tion in the chromosome binding motif to provide higher on-target fidel-
.. ity without loss of cleavage efficiency. The ratio between Cas9 and
..
CRISPR/Cas9 presents many advantages compared to previous technol- .. sgRNA116,117 can also influence the occurrence of off-targets. By reduc-
ogies, but also comes with some limitations and issues including variabil-
.. ing the number of Cas9 compared to the number of sgRNA, the on-
..
ity in its efficiency and potential off-target gene editing. In addition, .. target events are promoted.113 On the contrary, if the proportion of
germline editing using CRISPR/Cas9, especially in humans, raise societal,
.. Cas9 is too high, the number of off-target events increases.117,118
..
and ethical considerations.47 .. Even if these measures have been shown to prevent and greatly re-
.. duce the occurrence of off-targets events, different screening methods
..
.. are applied to detect the genome editing outcomes and have been ex-
4.1 Variable efficiency .. tensively reviewed elsewhere.119 These methods have been either de-
Despite progressive optimization of the tools, the CRISPR/Cas9 system ..
.. veloped to detect mutations in pre-selected regions or on a genome-
presents variable efficiency which could stem from several parameters .. wide scale. In addition to exome- and whole-genome sequencing, some
linked to the sgRNA/DNA binding, NHEJ/HDR frequency, and the deliv- ..
.. methods can specifically detect DSBs caused by nuclease activity such as
ery to the targeted organism. .. 120 121
In addition to its influence on DNA repair mechanisms, the cell cycle
.. GUIDE-seq or Digenome-seq. GUIDE-seq is based on the integra-
.. tion of double-stranded oligodeoxynucleotides into DSBs after NHEJ
is suspected to influence gene editing outcomes. Indeed, the chromatin ..
structure depends on the phase of the cell cycle and a compacted chro-
.. and sequencing of the tagged fragments.120 Digenome-seq is based on
.. the whole-genome sequencing of DNA fragments generated after in vitro
matin can limit the access of sgRNA and Cas9 to the targeted DNA.104 ..
Recent reports have identified Cas9 affinity to the target DNA as an im-
.. digestion with Cas9 and sgRNAs.121 The later technique is amenable to
.. multiplexing and can be used to analyse up to 10 sgRNAs in one se-
portant factor that may impact the system effectiveness.105 However, us- ..
ing a tracking system to visualize Cas9 target searching, it was shown that
.. quencing run. These two techniques have been largely used for the as-
.. sessment of off-target events following in vitro genome editing, but are
Cas9 interrogates mammalian genomes through diffusion with slower ..
.. more difficult to implement for in vivo genome editing experiments. In a
movement within heterochromatin.106 .. recent study,122 a full strategy for the verification of in vivo off-targets (so
It has been shown that DSBs induced by Cas9 cause a p53-mediated ..
.. called VIVO) was proposed and combined an initial ‘discovery’ step of
toxic response that inhibits CRISPR–Cas9 action.107 Further, it has been .. in vitro off-target sites using CIRCLE-seq,123 followed by a ‘confirmation’
reported that inhibition of p53 prevents the damage response to DSBs ..
.. step looking for the presence of the previously identified off-target sites
and increases HDR from a DNA template.108 The later results suggest .. in vivo.122 This strategy was applied to perform PSCK9 genome editing
that CRISPR/Cas9 genome editing should be more efficient in cells with ..
.. in vivo in mice liver, and was demonstrated as a robust and sensitive
low p53 protein activity, a phenotype common to tumour cells. .. method to detect off-target mutations with frequencies as low as
Whether CRISPR/Cas9 can be more efficient in cells with a tumour po- ..
.. 0.13%.122
tential is currently debated.107,108 ..
Lastly, the PAM sequence is a constraining factor for CRISPR/Cas9 ef- ..
.. 4.3 Immunogenicity concerns
ficiency, as it is mandatory for Cas9 DNA docking. The presence or ab- .. One of the most widely used Cas9 is derived from the bacteria S. pyo-
sence of a PAM sequence in the downstream sequence of the targeted ..
.. genes, a common cause for infection in human and a source of a pre-
DNA region is a limiting factor, but Cas9 orthologues have been ..
reported thus covering a larger panel of PAM sequences.109 The PAM .. existing immune response against the Cas9 homologue.124 Similarly, it
.. has been demonstrated the existence of a pre-existing immune response
sequence can also be masked by nucleosomes,104,110 thereby impacting ..
the correct recognition between PAM and Cas9 and limiting DNA break
.. to the St. aureus Cas9 homologue.125 To assess the potential host-
.. response to bacteria-derived Cas9 proteins, a long-term evaluation of
initiation by Cas9. ..
.. mdx mice treated with AAV-Cas9 (to excise the pathogenic exon 23)
.. was recently reported.92 AAV-Cas9 was demonstrated as immunogenic
..
4.2 Off-target events .. in almost all the treated animals when administered at the adult age
One of the most challenging and developed issue with CRISPR/Cas9 .. (8 weeks old). The humoral and cellular immune responses were how-
..
technology is the occurrence of off-target editing events. While the .. ever avoided by treating neonatal mice (Day 2 after birth). There is a the-
CRISPR/Cas9 system is an accurate DNA targeting mechanism, off- .. oretical concern that Cas9-reactive T cells may alter the genome editing
..
targets refer to the Cas9 action on an unwanted site with sequence ho- .. efficiency to treat disease in vivo by killing the Cas9-expressing cells and
mology.111 This supposes a tolerance of Cas9 for mismatches between .. may even result in a more significant immune response creating major
..
the guide and the targeted sequences. Advanced design tools of .. safety concerns. However, in this study,92 the genome editing events
sgRNA25 help in predicting off-target sites and defining priority sequence .. were sustained for 1 year and were not influenced by the occurrence of
..
to inspect for a quality control.112 Several analysis tools have been devel- .. the immune response against the bacteria-derived Cas9. This important
oped to anticipate and reduce the probability of off-target activity.24,25
.. study indicates that the immune response to AAV-Cas9 must be consid-
..
Moreover, it has been shown that using a truncated RNA guide, . ered during future clinical developments of in vivo genome editing. As for
..
previous gene therapy trials, it remains to be determined if this response .. in vivo. There are multiple applications in cardiovascular medicine, with a
can be avoided with a transient immunosuppression protocol, or if fur- .. primary focus on direct therapeutic interventions to treat inherited car-
..
ther in vivo applications would require the development of a non- .. diac disorders. As explained in this review, the development of in vivo so-
immunogenic Cas9. .. matic genome editing is hampered by different factors including
..
.. difficulties to deliver the genome editing tools to the cardiac muscle, low
4.4 Ethics .. rates of homology recombination DNA repair in non-dividing cells (such
..
The potential of the CRISPR/Cas9 technology finally comes with impor- .. as cardiomyocytes) and potential immune responses to bacteria-derived
.. Cas9 proteins when administered in adult organisms. Alternative tools
tant societal and ethical issues. As the technology can allow manipulation ..
of almost any DNA sequence, it raises concerns of how ethical and ac- .. such as base editors could represent a powerful option, but the optimal
.. conditions leading to an efficient and safe genome editing in vivo (and in
ceptable by the society is a specific genetic manipulation. The most evi- ..
dent ethical issue concerns the manipulation of germ cells rather than .. the cardiac muscle) remains to be defined. In contrast, germline genome
.. editing seems to be more streamlined and practicable but can raise ethi-
somatic cells. From a genetic viewpoint, correcting a mutation at the ..
germ cell level is a way to directly treat the disease and avoid its trans- .. cal questions, notably when proposed in human embryos. Even if some
.. studies are required to better match the basics of CRISPR/Cas9 genome
mission to future generations but reciprocally all genome editing events ..
will be transmitted to the future generations. Over the long-term, this .. editing with cardiovascular specificities, the world of genome editing has
.. been remarkably characterized by a fast-paced technological growth
strategy could offer a way to cure genetic mutations associated with ter- ..
ribly disabling diseases. As the description of the human genome has .. allowing now what was unimaginable few years ago. Beyond these tech-
.. nological developments, there is now a predominant concern on how
allowed the identification of multiple disease-related mutations, CRISPR/ ..
Cas9 now offers opportunities to develop targeted therapies and elimi- .. the scientific community, including in the cardiovascular field, would gov-
.. ern and guide the future development and applications of these
nate them. However, the clinical expression of disease-causing mutations ..
can be variable as a consequence of incomplete penetrance, the pres-
.. techniques.
..
ence of modifier genetic variants, or the association with specific envi- .. Conflict of interest: none declared.
ronmental stressors.126
..
..
For example, a CRISPR/Cas9 strategy was recently developed to cor- ..
rect in human embryos a MYBPC3 mutation associated with the onset of
..
.. Funding
HCM.71,82 HCM causes a broad spectrum of clinical manifestations with ..
a high risk of cardiac arrhythmias and sudden death in its more unfavour-
.. This work was supported by Inserm, Association Nationale de la Recherche
.. et de la Technologie (ANRT, Cifre2017/1406), ANR (grant 17-CE17-0015-
able forms. However, multiple patients present limited symptoms and ..
.. 02) and Fondation Leducq (grant 18CVD05).
good prognosis, and the clinical manifestation of MYBPC3 mutations are ..
often delayed until middle or old age.127 This shows that the use of ..
..
CRISPR/Cas9 in the germline is not limited by a technical issue but must .. References
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Artículo CRISPR

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Cardiovascular Research (2020) 116, 894–907 REVIEW

CRISPR/Cas9 gene-editing strategies in

.. DNA manipulation in yeast3 it has become increasingly feasible during

Table 1 Characteristics of main Cas enzymes

main approaches developed thus far. A first approach is based on gene

CRISPR/Cas9 represents a breakthrough advance in genome editing

14. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero

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