. 2023 Mar 9;11:1143157. doi: 10.3389/fbioe.2023.1143157

TABLE 2.

Strategies to reduce off-target effects.

	Classification	Description	Effect
Cas9 improvement	SpCas9 variants [65–69]	SpCas9-HF1, eSpCas9, hypaCas9: rational designed SpCas9 mutants	Maintained or increased on-target efficiency and decreased off-target effects
		EvoCas9: unbiased high-throughput screen
		Paired SpCas9 nickase: mutate at RuvC or HNH domain
	Cas9 orthologous [70–71]	SaCas9: from Staphylococcus aureus; (PAM: 5’-NGGRRT-3’)	Complex PAM sequence
	Cas9 orthologous [70–71]	St1Cas9, St3Cas9: From Streptococcus thermophilus; (PAM: 5’-NNAGAAW-3’ and 5’-NGGNG-3’)	Complex PAM sequence
gRNA improvement	gRNA length adjustment [72–75]	Extended gRNA: GGX20	Decreased off-target effects
	gRNA length adjustment [72–75]	Truncated gRNA: truncated by 2-3bp at 5’ end	Decreased off-target effects
	Chemical modifications [76–78]	Incorporation of MP/bridged nucleic acids/locked nucleic acids	Increased on-target efficiency and decreased off-target effects
	Chemical modifications [76–78]	Replacement of RNA nucleotides into DNA nucleotides
Delivery methods improvement	Application in cell culture [89–94]	Plasmid transfection	May cause accumulation of off-target mutations
		Viral transduction	May cause accumulation of off-target mutations
		RNP electroporation: Rapid transfection and turnover	Eliminate off-target effects
	Application in vivo [95–105]	AAV: Last for years in terminally differentiated cell types	May cause accumulation of off-target mutations
	Application in vivo [95–105]	LNP: Can be quickly degraded in vivo	Eliminate off-target effects