Table 1.

CRISPR/Cas9 improved variants.

Name	Key Features	Reference
Cas9 variants engineered using random mutagenesis combined with high-throughput screening
Sniper-Cas9	Sniper-Cas9 showed much higher on-target activities and comparable specificity at most off-target loci when compared with other engineered high-fidelity SpCas9 variants such as SpCas9-HF1, HypaCas9, evoCas9, and eSpCas9(1.1). However, Sniper-Cas9 showed stronger tolerance to single mismatches in the PAM-distal region (e.g., 16th, 18th, and 19th). Sniper-Cas9 showed on-target activities with extended or truncated sgRNAs and worked well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing	[33]
HiFi Cas9	HiFi Cas9 (R691A variant) demonstrated the highest on-target activity (82%) in the RNP format compared to other improved Cas9 variants	[30]
xCas9	Cas9 variant with expanded PAM compatibility (xCas9 can recognize a broad range of PAM sequences, including NG, GAA, and GAT). xCas9 demonstrated lower genome-wide off-target activity at all NGG target sites, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs	[38]
SpartaCas	SpartaCas (D23A, T67L, Y128V, and D1251G variants) had reduced off-target events while maintaining high on-target editing in T-cells. The editing efficiency of SpartaCas was slightly reduced when compared to wild-type Cas9, but it demonstrated dramatically higher editing efficiency when compared to SpCas9-HF4 and eCas9	[34]
efSaCas9	efSaCas9 (N260D variant) is a high-fidelity, high-activity variant that could be harnessed for safe and reliable genome editing. efSaCas9 demonstrated dramatically reduced off-target effects (approximately 2- to 93-fold improvements) compared to wild-type SaCas9	[28]
Cas9 variants engineered using a structure- and/or function-guided protein engineering approach
SpCas9-D1135E	SpCas9-D1135E possessed genome-wide specificity and demonstrated reduced off-target effects. The D1135E mutant was able to better discriminate between NGG and NGA PAMs compared with wild-type SpCas9. Moreover, it had decreased activity against non-canonical NAG, NGA, and NNGG PAMs relative to wild-type SpCas9	[25]
eSpCas9	eSpCas9 variants, also known as “enhanced specificity”, SpCas9 showed reduced off-target effects and maintained robust on-target cleavage.	[27]
SpCas9-HF1 (and -HF2, -HF3, -HF4)	SpCas9-HF are high-fidelity SpCas9 variants harboring alterations designed to reduce non-specific DNA contacts. SpCas9-HF retained on-target activities comparable to wild-type. SpCas9-HF rendered all or nearly all off-target events undetectable, even for atypical, repetitive target sites	[24]
HypaCas9	HypaCas9 (N692A, M694A, Q695A, and H698A variants) exhibited dramatically improved genome-wide specificity compared to wild-type SpCas9 and showed equivalent or better genome-wide specificity relative to both SpCas9-HF1 and eSpCas9 variants	[31]
SuperFi-Cas9	SuperFi-Cas9 was able to discriminate between on- and off-target substrates without compromising DNA cleavage efficiency, but a recent study showed that it had significantly reduced on-target activity in mammalian cells	[36,84]
LZ3 Cas9	The LZ3 Cas9 variant is known to exhibit high activity, increased specificity, and a differential +1 insertion profile as compared to wild-type SpCas9. Further rational engineering of LZ3 Cas9 might provide novel opportunities for non-templated correction of disease-causing frameshift mutations in the human population	[37]
eSaCas9	eSaCas9 is a high-fidelity version of SaCas9 obtained by weakening interactions between Cas9 and the target DNA. eSaCas9 demonstrated high on-target activities and comparable specificity at most off-target loci	[27]
SaCas9-HF	SaCas9-HF showed high genome-wide targeting accuracy without compromising on-target efficiency, as validated by rigorous evaluation of its on- and off-target activities across multiple endogenous sites	[32]
KKH-SaCas9-SAV1 (and -SAV2)	KKH-SaCas9-SAV1 and SAV2 are SaCas9 variants that exhibited low off-target and high on-target activities and revealed a pivotal role of the previously unreported Y239H substitution in determining target accuracy while maintaining the activity of KKH-SaCas9	[39]
Cas9 variants engineered using directed evolution combined with structure-guided modeling
evoCas9	EvoCas9 is a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (4-fold average improvement), while maintaining near-wild-type on-target editing efficiency (90% median residual activity)	[29]
SaCas9-Q414A	The Q414A variant of SaCas9 exhibited even higher fidelity than the N260D variant of SaCas9 while retaining the most on-target activity	[28]
Cas9-based fusion proteins with improved properties
miCas9	Cas9 variant with improved homology-directed repairing capacity (2.5-fold higher). To improve Cas9’s homology-directed repair capacity, SpCas9 was fused to a minimal motif consisting of thirty-six amino acids (Brex27 motif)	[40]
Cas9-pDBD	Fusing a programmable DNA-binding domain (pDBD) to Cas9 combined with the attenuation of Cas9’s inherent DNA binding affinity produced a Cas9-pDBD chimera with dramatically improved precision and increased targeting range	[85]
Cas-Cas9 chimeras	RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, were designed as versatile genome engineering systems. In both formats, Cas9-Cas9 fusions displayed an expanded targeting repertoire and achieved highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras had higher target site activity and generated predictable, precise deletion products between target sites	[86]