SnapShot: Class 1CRISPR-Cas Systems

Kira S. Makarova,1 Feng Zhang,2,3 and Eugene V. Koonin1

1
National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA;
2
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 3McGovern Institute for Brain Research,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Effector complex Classification Effector complexes
cas6 Generalized organization
DNA Archaeoglobus fulgidus
I-A
AF1859,AF1870-AF1879 Repeat region (crRNA)
SS (cas11) cas7 cas5 cas8a1 cas3 cas3 cas2 cas4 cas1 cas4 CRISPR RAMP
csa5 (repeat
DNA Clostridium kluyveri
Cas7 Cas6 Group 6 associated
RAMP

Group 7 RAMP
I-B
CKL_2758-CKL_2751 Cas7
mysterious
cas6 cas8b1 cas7 cas5 cas3 cas4 cas1 cas2 CRISPR Small proteins)
subunits superfamily
Cas7
DNA Bacillus halodurans
I-C BH0336-BH0342 Spacer region Three
cas3 cas5 cas8c cas7 cas4 cas1 cas2 CRISPR Cas7 (crRNA) distinct

Large subunit
groups:
GSU0054 Geobacter Cas7
DNA sulfurreducens Cas5
I-U Seed region
Cas6

Group 5
GSU0051-GSU0054 (crRNA)
I

RAMP
cas3 cas8u2 cas7 cas5 cas6 cas4 cas1 cas2 CRISPR GSU0057-GSU0058 Cas7
Cas5 5 handle region
DNA cas3 (crRNA)
Cyanothece sp. 8802
I-D Cyan8802_0527- Cas8/Cas10
cas3 cas10d cas7 cas5 cas6 cas4 cas1 cas2 CRISPR Cyan8802_0520
csc2 csc1 RRM fold RRM fold Cas6
DNA Escherichia coli K12 effector proteins (Typical RAMP)
I-E Beta Alpha
ygcB-ygbF Cas6
cas3 cas8e SS (cas11) cas7 cas5 cas6 cas1 cas2 CRISPR strand helix RRM fold
cse1 cse2 pdb: 3I4H
Yersinia pseudo- Cas10 2
DNA 3
I-F tuberculosis 1
YPK_1644-YPK_1649 4 1 3 2 G-rich loop 4
cas1 cas2 cas3 cas8f cas5 cas7 cas6f CRISPR 41 2
csy1 csy2 csy3 3
IV Acidithiobacillus C N
Cas5
IV ferrooxidans Cas6
AFE_1037-AFE_1040 Cas7 Catalytic
dinG cas8-like cas7 cas5 G-rich
csf4 csf1 csf2 csf3 residues loop
DNA Staphylococcus
III-A epidermidis
RNA
cas6 cas10 SS (cas11) cas7 cas5 cas7 csm6 cas1 cas2 CRISPR
SERP2463-SERP2455 Origin
csm2 csm3 csm4 csm5 RRM2 RRM1 Helical
DNA? Synechocystis sp. 6803
III-D sll7067-sll7063 HD Ancestral innate
RNA? cas10-like immunity system
cas10 cas7 cas5 SS (cas11) cas7 cas7 cas7
csm3 csx10 csm2 csm5 csm5 all1473 csm5 • Insertion of casposon
III DNA? Methanothermobacter next to cas10-like gene
III-C thermautotrophicus Toxin-antitoxin cas2
• CRISPR repeats evolved
RNA? cas7 cas7 cas10 MTH328-MTH323 system
cas7 SS (cas11) cas5 from terminal repeats
cmr1 cmr6 cmr4 cmr5 cmr3 of casposon
Terminal repeats
DNA • Cas1 originated from
III-B Pyrococcus furiosus the same casposon
RNA PF1131-PF1124 cas1
cas7 cas10 cas5 cas7 SS (cas11) cas6 cas7 cas1 cas2 • Acquisition of Cas2-like toxin
cmr1 cmr3 cmr4 cmr5 cmr6 from toxin-antitoxin system
Adaptation
module
cas2
Large Mechanism of action
subunit cas7 cas6 cmr3 cas6 cas1
Ancestral adaptive
cas10 immunity system
Type I Type III CRISPR cas1
• Duplication of cas10-like
cas3 Small cas5 cas1 cas2 CRISPR cmr1 cas10 Small cmr4 cmr6 cas2 CRISPR gene followed by its fission
subunit subunit • Origin of cas7 family and
Adaptation crRNA maturation Targeting DNA small subunit (SS)
and loading Invading
Type I and III Effector Type I DNA Type III cas1
cas10 cas7
complex Type I and III RNA polymerase
• Origin of G-rich loop
Cas3 3 5 and duplication of
DNA R-loop 5
phage Priming Small Cas7-like protein
cas6 3 3 CRISPR cas2
effect HD subunit • Origin of RAMP
Invading Proto- Cas1 pre-crRNA domain (SS) containing one
DNA PAM
spacer
Cas2
I-C RRM domain
5 with G-rich loop
pre-crRNA Cas3 ssDNA
3 cas1 G-rich loop
Cas4 5 3 G-rich loop
5
Cas5 HD nuclease SS
crRNA
Some III-B 5
mRNA
CRISPR cas2
cas10 cas7 RAMP
CRISPR 3 3
5 5 Cyclase • Duplication of RAMP and
RNA phage Reverse 3 3
trancriptase domain origin of cas5 family
5 GGDD cas1
Targeting RNA active site
Proto-
Invading spacer Type I Type III Type III 3
SS
RNA Cas2 3 CRISPR cas2 cas10 cas7 cas5
Cas1 effector effector
complex complex Invading 5 • Duplication of cas5 and
RNA Cmr4 origin of cas6 family
CRISPR 5 or Csm3
cas1 Ancestral
Effector module adaptive
Ancillary proteins immunity
Class 1
cas10 cas7 cas5 SS cas6
Cas2, RNA nuclease CARF (CRISPR-associated Rossmann fold) Reverse transcriptase (RT) CRISPR cas2 system
SSO1404 Ancestral Type III system
Cas2 Sulfolobus CARF HEPN PDB: 4EOG/2I71, PF1127 RT
Csx1 alr1468
solfataricus HTH Pyrococcus furiosus Cas1 Nostoc sp. • Duplication of Cas7
pfam09455 cas3 cas1
PDB: 5FSH, Rv2818c
Cas2 Cas3 y1723 Csm6 6H Mycobacterium Cas6 Cas1 Psta_1137
Yersinia pestis pfam09659 Pirellula staleyi
tuberculosis cas10 cas7 cas5 SS cas6 cas7
CRISPR cas2
LSEI_0356 TTHB152 Other families
Cas2 Lactobacillus pfam09670 Thermus thermophilus WYL domain and HTH Ancestral Type I system
casei aq_376 Csx15 TTE2665
Aquifex aeolicus sll7009 • Origin of Cas8 by
TM Synecho- Thermoan-
RelE aerobacter inactivation of Cas10
Cas4, DNA nuclease sll7062 cystis sp. • Acquisition of Cas3 helicase
Synechocystis sp. tengcongensis
PIN
Csx3, RNA nuclease • HD domain transferred from
COG1468 PAE0079 ST0035 Csx18 Cas10 to Cas3
Pyrobaculum pfam09651 aq_377 Ple7327_2251 cas1
Cas4 Sulfolobus tokodaii Aquifex
aerophilum Pleurocapsa sp.
PDB: 3QYF, Sso1393 aeolicus
COG4343 PAE0082 Sulfolobus solfataricus Csx20 MJ_1673
Csx16 VVA1548 Methano- cas3 cas8 cas5 cas7 SS cas6
Cas4a Pyrobaculum CRISPR cas2
aerophilum PDB: 1XMX, Vc1899 Vibrio caldococcus
pfam09002 Vibrio Cholerae vulnificus jannaschii Ancestral Type IV system
NE0113 PRIMPOL
GSU0057 pfam09623 family protein Sfum_1354,Sfum_1355 • Reduction of large subunit (LS)
Cas1 Cas4 Geobacter Nitrosomonas europaea
sulfurreducens PDB: 2WTE, SSO1445 Cas1 Syntrophobacter CRISPR LS SS cas7 cas5
• Loss of adaptation module
Csa3 Sulfolobus solfataricus fumaroxidans and Cas6

See online version for

946 Cell 168, February 23, 2017 Published by Elsevier Inc.  DOI http://dx.doi.org/10.1016/j.cell.2017.02.018 legends and references
SnapShot: Class 1 CRISPR-Cas Systems
Kira S. Makarova,1 Feng Zhang,2,3 and Eugene V. Koonin1
1
National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA;
2
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 3McGovern Institute for Brain Research,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Classification
CRISPR-Cas adaptive immune systems of prokaryotes split into two distinct classes based on effector module organization. Class 1 CRISPR-Cas systems utilize multi-protein
effector complexes, whereas class 2 CRISPR-Cas systems utilize single-protein effectors (Makarova et al., 2015). Primarily based on distinct effector protein families, Class 1
systems are divided into 3 types and 12 subtypes (Makarova et al., 2015). Class 1 systems represent about 90% of the CRISPR-Cas loci and are found in diverse bacterial and
archaeal phyla; type III systems are strongly enriched in thermophiles (Makarova et al., 2015). In addition to the effector genes, most of the class 1 loci encode adaptation module
proteins, Cas1 and Cas2, and multiple accessory proteins, such as Cas4, reverse transcriptase, CARF (CRISPR-associated Rossmann fold) domain-containing protein, and
others. Type III and type IV systems often lack adaptation module genes and/or CRISPR arrays in their respective loci. All type I systems also encode DNA helicase Cas3, which
is often fused to an HD family nuclease domain. The repeats of class 1 systems are usually palindromic. In type I systems, a protospacer adjacent motif (PAM), which varies
between subtypes and is located either 5′ or 3′ of the (proto)spacer, is required for both adaptation and interference (Leenay et al., 2016).

Mechanism of Action
Spacer acquisition during adaptation, the process of acquiring new immune memories by CRISPR systems, requires Cas1 and Cas2 proteins; the nuclease activity of Cas1,
but not that of Cas2, is essential. In addition, involvement of Cas3, Cas4, and the effector complex in adaptation has been demonstrated for type I systems (Mohanraju et al.,
2016). For some of the III-B systems, acquisition of spacers originating from RNA and the role of reverse transcriptase fused to Cas1 protein in this process have been demon-
strated (Silas et al., 2016). Pre-crRNA is usually processed by the dedicated ribonuclease Cas6, which cleaves the repeat to produce a CRISPR (cr)RNA with a 5′ handle, spacer,
and 3′ stem-loop structure. In I-C systems, this function is performed by Cas5, which is both a ribonuclease and an effector complex subunit (Mohanraju et al., 2016). The same
crRNAs can be shared by different type I or type III complexes that are present in the same cell. The Cas7 proteins function as a “ruler,” binding the spacer region to form a chain
with a varying number of monomers depending on the length of the spacer (Mohanraju et al., 2016). The dedicated interference helicase-nuclease Cas3 is recruited by the type I
effector complexes upon recognition of the PAM and a cognate protospacer and R-loop formation. The HD domain is responsible for DNA cleavage. Type III systems lack Cas3
and do not recognize a PAM. In these systems, DNA cleavage requires the activities of both the HD domain and the cyclase domain of Cas10, the large subunit of the effector
complex (Mohanraju et al., 2016). Type III systems can also target ssRNA through the Cas7 family proteins, such as Csm3 in subtype III-A and Cmr4 in subtype III-B. The RNA
and DNA degradation reactions in these systems are coupled, ensuring targeting of actively transcribed phage DNA (Kazlauskiene et al., 2016).

Effector Complex Architecture

Despite extremely low or undetectable sequence similarity, type I and III systems share common principles of effector complex organization (Makarova et al., 2011). Several
cryo-electron microscopy structures for both type I and III systems effector complexes show remarkable similarity of the overall architecture and shape. Most of the subunits
belong to the RAMP (repeat associated mysterious proteins) family that consists of three distinct groups (Cas5, Cas6, and Cas7). Typically, Cas6 and Cas5 proteins contain two
RRM (RNA recognition motif) domains, whereas Cas7 is a single-RRM protein. Effector complexes contain one Cas5 subunit and several Cas7 subunits. The Cas5 subunit binds
the 5′ handle of the crRNA and interacts with the large subunit of the effector complex (Cas8 in type I and Cas10 in type III), forming a stable, two-subunit subcomplex. The small
subunit is typically present in several copies and interacts with the crRNA backbone bound to Cas7 (Mohanraju et al., 2016).

Regulatory and Other Ancillary Functions

Several groups of Cas proteins that are common in Class 1 systems so far have not been linked to a specific function. In particular, protein containing the CARF domain that is
predicted to bind nucleotide ligands and is typically fused to a DNA-binding domain and different nuclease domains is implicated in the regulation of the CRISPR-Cas function,
including sensing genotoxic stress and possibly coupling adaptive immune response to programmed cell death (Makarova et al., 2014).

Origin and Evolution

The Class 1 effector complexes center around the RRM domains that are present in Cas10 and all three families of RAMPs. Considering also the homology between the
C-terminal domain of the large subunit (Cas8 and Cas10 in types I and III, respectively) and the small subunit of the effector complexes, these complexes might have evolved
from a single Cas10-like protein containing an RRM domain with nuclease activity (Shmakov et al., 2015). Thus, the ancestral class 1 systems could have most closely resembled
modern type III. Cas1 and CRISPR repeats probably originated from transposable elements, known as Casposons, that utilize Cas1 homologs as integrases (Koonin and Krupo-
vic, 2015). Loss of the catalytic activity of the large subunit and recruitment of Cas3 for DNA cleavage were the key events in the evolution of type I systems. Further degradation
of the large subunit and loss of the adaptation module (Cas1 and Cas2) yielded type IV systems.

Anti-CRISPR Proteins and Bacteriophage-Encoded CRISPR-Cas Systems

At least some bacteriophages encode multiple anti-CRISPR proteins (Pawluk et al., 2016). Although such proteins have been experimentally studied only in Pseudomonas
phages and shown to inhibit I-F systems, it can be predicted that other viruses and integrated elements encode numerous currently uncharacterized proteins that act against
most of CRISPR-Cas systems. Conversely, some bacteriophages have been shown to encode type I CRISPR-Cas systems that target host defense genes (Mohanraju et al.,
2016).

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946.e1 Cell 168, February 23, 2017 Published by Elsevier Inc.  DOI http://dx.doi.org/10.1016/j.cell.2017.02.018