[The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory]

Mol Biol (Mosk). 2024 Jan-Feb;58(1):157-159.
[Article in Russian]

Abstract

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

Keywords: HH theory; SpCas9; cleavage efficiency; off-target; optimization; variants.

Publication types

  • English Abstract

MeSH terms

  • CRISPR-Associated Protein 9* / genetics
  • CRISPR-Associated Protein 9* / metabolism
  • CRISPR-Cas Systems*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • Gene Editing* / methods
  • HEK293 Cells
  • Humans
  • INDEL Mutation
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Streptococcus pyogenes* / enzymology
  • Streptococcus pyogenes* / genetics

Substances

  • CRISPR-Associated Protein 9
  • RNA, Guide, CRISPR-Cas Systems
  • DNA