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Summary

Researchers screened SpCas9 orthologs for genome editing potential. Ten showed activity in human cells, with some offering enhanced specificity and superior base editing efficiency, like SeqCas9.

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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.