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Compact SchCas9 Recognizes the Simple NNGR PAM.

Shuai Wang1, Huilin Mao1, Linghui Hou1

  • 1State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, 200438, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|December 7, 2021
PubMed
Summary
This summary is machine-generated.

Researchers expanded genome editing tools by discovering new CRISPR/SaCas9 variants with diverse PAMs. SchCas9 shows the most relaxed PAM, enabling efficient editing in human cells for research and clinical use.

Keywords:
CRISPR/Cas9PAMSaCas9SchCas9genome editing

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Clustered regularly interspaced short palindromic repeat (CRISPR)/Staphylococcus aureus Cas9 (SaCas9) is a widely used in vivo genome editing tool.
  • Previous work established four SaCas9 orthologs for genome editing applications.

Purpose of the Study:

  • To broaden the targeting capabilities of SaCas9 by exploring the diversity of its protospacer adjacent motifs (PAMs).
  • To identify novel SaCas9 orthologs with distinct PAM specificities for enhanced genome editing applications.

Main Methods:

  • Screening of 16 SaCas9 orthologs to assess their genome editing activity in mammalian cells.
  • Identification and characterization of protospacer adjacent motif (PAM) sequences recognized by active SaCas9 orthologs.

Main Results:

  • Twelve of the 16 screened SaCas9 orthologs demonstrated editing activity in mammalian cells.
  • Five distinct PAM types were identified: NNGRRT, NNGRRR, NNGRC, NNGA, and NNGR.
  • The Staphylococcus capitis Cas9 (SchCas9) ortholog exhibited the most relaxed PAM preference to date (NNGR) among compact Cas9 variants.
  • SchCas9 facilitated efficient genome editing across multiple human cell lines.

Conclusions:

  • The study introduces a panel of compact Cas9 tools with expanded targeting scopes and diverse PAM specificities.
  • SchCas9 represents a valuable new option for genome editing in basic research and clinical settings due to its relaxed PAM recognition.
  • These findings contribute to the development of more versatile and efficient genome editing technologies.