Enhanced genome editing with a Streptococcus equinus Cas9

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Numerous CRISPR-associated protein 9 (SpCas9) orthologs have been identified computationally.
• The genome editing capabilities and specificities of these SpCas9 orthologs are largely unexplored.
• Understanding SpCas9 ortholog diversity is crucial for advancing gene editing technologies.

Purpose of the Study:

• To screen a panel of SpCas9 orthologs for genome editing activity and specificity in human cells.
• To identify novel SpCas9 variants with improved properties for gene editing applications.
• To evaluate the potential of these orthologs, particularly SeqCas9, for base editing.

Main Methods:

• A green fluorescent protein (GFP)-activation assay was employed to screen 18 SpCas9 orthologs.
• Assays were conducted in human cells to assess genome editing activity.
• Specificity was evaluated, and base editing efficiency was compared at endogenous loci.

Main Results:

• Ten out of 18 SpCas9 orthologs exhibited detectable genome editing activity in human cells.
• Several orthologs demonstrated a preference for purine-rich protospacer adjacent motif (PAM) sequences.
• Four orthologs showed improved specificity over the wild-type SpCas9.
• SeqCas9, recognizing an NNG PAM, displayed activity and specificity comparable to SpCas9-HF1.
• SeqCas9 demonstrated enhanced base editing efficiency compared to SpCas9-NG and SpCas9-NRRH.

Conclusions:

• The study highlights the significant, yet largely untapped, potential of SpCas9 orthologs for genome editing.
• Specific SpCas9 variants, such as SeqCas9, offer advantages in terms of specificity and base editing efficiency.
• These findings expand the toolkit for precise and efficient genome editing, particularly for base editing applications.