. Author manuscript; available in PMC: 2020 Aug 1.

Published in final edited form as: Nat Rev Mol Cell Biol. 2019 Aug;20(8):490–507. doi: 10.1038/s41580-019-0131-5

Table 1.

Cas9 variants with altered PAM and targeting specificities

Name	Included mutations	PAM (5’ to 3’)	Notes
SpCas9	Native Streptococcus pyogenes Cas9	NGG²⁴⁹	1368 amino acids
VRER SpCas9	D1135V, G1218R, R1335E, T1337R	NGCG¹⁷	Altered PAM variant; Bacterial-selection-based screening
VQR SpCas9	D1135V, R1335Q, T1337R	NGAN or NGNG¹⁷	Altered PAM variant; Bacterial-selection-based screening
EQR SpCas9	D1135E, R1335Q, T1337R	NGAG¹⁷	Altered PAM variant; Bacterial-selection-based screening
xCas9-3.7	A262T, R324L, S409I, E480K, E543D, M694I, E1219V	NG, GAA, GAT¹⁸	Altered PAM variant; Phage-assisted continuous evolution
eSpCas9 (1.0)	K810A, K1003A, R1060A	NGG	Enhanced specificity; Structure-guided protein engineering¹⁹
eSpCas9 (1.1)	K810A, K1003A, R1060A	NGG	Enhanced specificity; Structure-guided protein engineering¹⁹
Cas9-HF1	N497A, R661A, Q695A, Q926A	NGG	Enhanced specificity²⁰
HypaCas9	N692A, M694A, Q695A, H698A	NGG	Enhanced specificity²¹
evoCas9	M495V, Y515N, K526E, R661Q	NGG	Enhanced specificity; yeast-based screening²²
HiFi Cas9	R691A	NGG	Enhanced specificity for ribonucleoprotein delivery²³
ScCas9	Native Streptococcus canis Cas9	NNG²⁵⁰	1375 amino acids
StCas9	Native Streptococcus thermophilus Cas9	NNAGAAW^11,25	1121 amino acids
NmCas9	Native Neisseria meningitidis Cas9	NNNNGATT^26-28	1082 amino acids
SaCas9	Native Staphylococcus aureus Cas9	NNGRRT²⁹	1053 amino acids
CjCas9	Native Campylobacter jejuni Cas9	NNNVRYM³⁰	984 amino acids
CasX	Deltaproteobacteria and Planctomycetes phyla	TTCN³²	980 amino acids

AAV, adeno-associated virus

ABA, abscisic acid

ABE, adenine base editor

ABI1, dimerizing ABA-binding protein

ADAR, adenosine deaminase acting on RNA

APEX2, engineered apurinic/apyrimidinic endodeoxyribonuclease 2 peroxidase

APOBEC1, apolipoprotein B mRNA editing enzyme complex-1

BE3, third generation base editor

BLESS, breaks labeling, enrichment on streptavidin and next-generation sequencing

Cas9-HF1, high fidelity Cas9 variant

CIB1, plant-derived cytochrome protein

CIRCLE-seq, circularization for in vitro reporting of cleavage effects by sequencing

CLOuD9, chromatin loop reorganization with CRISPR–dCas9

CORRECT, consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets

CRISPR, clustered regularly interspaced short palindromic repeats

CRISPRa, CRISPR activation

CRISPRi, CRISPR interference

crRNA, CRISPR RNA

CRY2, plant-derived cytochrome protein

dCas9, catalytically deficient Cas9

Digenome-seq, in vitro Cas9-digested whole-genome sequencing

DNA, deoxyribonucleic acid

DNMT, DNA methyltransferase

DSB, double-strand break

dsDNA, double-stranded DNA

enAsCas12a, enhanced AsCas12a variant

enCHIP, engineered DNA-binding molecule-mediated chromatin immunoprecipitation

EQR, Cas9 variant with triple mutations

eSpCas9, enhanced specificity SpCas9 variant

evoCas9, evolved high fidelity Cas9 variant

FKBP, rapamycin-binding domain

FRB, rapamycin-binding domain

GFP, green fluorescent protein

gRNA, guide RNA

GUIDE-seq, genome-wide, unbiased identification of DSBs enabled by sequencing

HCV, hepatitis C virus

HDR, homologous-directed repair

HEPN, higher eukaryotes and prokaryotes nucleotide binding domain

HiFi Cas9, high fidelity Cas9 variant

HITI, homology-independent targeted integration

HNH, Cas9 nuclease domain

HTGTS, high-throughput, genome-wide, translocation sequencing

HypaCas9, hyper-accurate Cas9 variant

Indels, insertions or deletions

iPSC, induced pluripotent stem cell

lncRNA, long non-coding RNA

KRAB, Krϋppel-associated box

LNP, lipid Nanoparticle

MEMOIR, mutagenesis with optical in situ readout

mRNA, messenger RNA

nCas9, Cas9 nickase

NHEJ, non-homologous end joining

p65, NF-κB transactivating subunit

PAM, protospacer adjacent motif

PAMmer, PAM-presenting oligonucleotide

PFS, protospacer flanking sequence

PYL1, dimerizing ABA-binding protein

REPAIR, RNA editing for programmable A to I replacement

RNA, ribonucleic acid

RNAi, RNA interference

RNP, ribonucleoprotein

RuvC, Cas9 nuclease domain

SHERLOCK, specific high sensitivity enzymatic reporter unlocking

smFISH, single-molecule RNA fluorescence hybridization

ssDNA, single-stranded DNA

ssODN, single-stranded oligodeoxynucleotide

stgRNA, self-targeting gRNA

TET1, ten-eleven translocation methylcytosine dioxygenase 1

tracrRNA, trans-activating crRNA

tRNA, transfer RNA

UGI, uracil glycosylase inhibitor

VIVO, verification of in vivo off-targets

VP64, four repeats of the herpes simplex VP16 activation domain

VQR, Cas9 variant with triple mutations

VRER, Cas9 variant with quadruple mutations

xCas9, expanded PAM SpCas9 variant

AsCas12a – Acidaminococcus spp. Cas12a

CjCas9 – Campylobacter jejuni Cas9

FnCas9 – Francisella novicida Cas9

LbCas12a – Lachnospiraceae spp. Cas12a

LwaCas13a – Leptotrichia wadei Cas13a

NmCas9 – Neisseria meningitidis Cas9

PaCsy4 – Pseudomonas aeruginosa Csy4

PspCas13b – Prevotella sp. P5-125 Cas13b

SaCas9 – Staphylococcus aureus Cas9

ScCas9 – Streptococcus canis Cas9

SpCas9 – Streptococcus pyogenes Cas9

StCas9 – Streptococcus thermophilus Cas9