Product Introduction：Alt-R S.p. HiFi Cas9 Nuclease V3 is also used for general genome editing, but it offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease V3 with the optimized Alt-R CRISPR-Cas9 gRNA (crRNA:tracrRNA) is highly recommended.

Increased specificity using Alt-R S.p. HiFi Cas9 Nuclease V3

As with the wild-type Alt-R Cas9 Nuclease V3, modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced on-target cleavage by Alt-R S.p. HiFi Cas9 Nuclease V3. However, Alt-R HiFi Cas9 Nuclease V3 also provides superior cutting specificity (minimized off-target editing).

Alt-R S.p. HiFi Cas9 Nuclease V3 facilitates near-WT on-target editing potency and reduces off-target site editing.RNP complexes were formed with either Alt-R S.p. Cas9 Nuclease V3 or Alt-R S.p. HiFi Cas9 Nuclease V3, combined with an Alt-R crRNA:tracrRNA complex targeting the EMX1 gene. RNP complexes (4 µM) were delivered into HEK-293 cells via nucleofection. Indel formation at the on-target locus as well as nine known off-target sites were measured by NGS (indicated on the y axis in log scale). N=1.