Table 1.

Strategies for engineering high-fidelity Cas9 proteins and representative variants.

Classification	Strategy	Representative High-Fidelity Cas9 Variants
Nonrational	Directed evolution (random mutagenesis plus high-throughput screening)	Sniper-Cas9 [23], HiFi Cas9 [20], xCas9 [30], SpartaCas [24], efSaCas9 [17]
Rational	Structure- and/or function-guided protein engineering	SpCas9 nickase [25], SpCas9-D1135E [27], eSpCas9 [18], SpCas9-HF1 (and -HF2, -HF3, -HF4) [26], HypaCas9 [21], SuperFi-Cas9 [28], LZ3 Cas9 [29], eSaCas9 [18], SaCas9-HF [22], KKH-SaCas9-SAV1 (and -SAV2) [31]
	Fusion protein	dCas9-FokI [33], miCas9 [32], Cas9-pDBD [59], Cas-Cas9 chimeras [60]
	Protein splitting	split-Cas9 [61]
Combined	Directed evolution plus structure-guided modeling	evoCas9 [19], SaCas9-Q414A [17]