VATIS UPDATE Article

Integrated DNA Technologies (IDT), the United States, has launched the first Cas9 enzyme variant that extensively reduces off-target effects in CRISPR ­genome editing without compromising on-target activity. The Alt-R® S.p. HiFi Cas9 Nuclease 3NLS enzyme is a recombinant S. pyogenes Cas9 mutant that improves specificity while maintaining a high editing efficiency similar to wild-type Cas9.

The launch represents a major step towards therapeutic use of CRISPR, which has previously borne the risk of the unwanted off-target editing events observed with wild-type Cas9. Earlier Cas9 mutants that offer improved specificity suffer from a moderate to severe loss of on-target activity when used as an RNP complex. CRISPR-Cas9 genome editing has revolutionized life science research, but concern about unwanted off-target editing events has been a stumbling block for researchers considering therapeutic applications.

The largely preferred method of delivering genome editing reagents as RNP complexes reduces, but does not eliminate, the risk of off-target editing. However, recent attempts at rational design of Cas9 mutants with reduced off-target activity traded on-target activity for improved specificity, and produced mutants generally unsuitable for use in RNP delivery. To successfully provide a Cas9 mutant with radically reduced off-target effects while maintaining high on-target activity, IDT screened more than 250,000 mutants in two rounds of selection.