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Can SpRY recognize any PAM in human cells?

Jinbin Ye1, Haitao Xi1, Yilu Chen1

  • 1Reproduction Center, Department of Obstetrics and Gynecology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, China.

Journal of Zhejiang University. Science. B
|May 13, 2022
PubMed
Summary

SpRY, a CRISPR-associated protein variant, expands genome editing capabilities. This study identifies its canonical and non-canonical protospacer adjacent motif sequences for precise human cell applications.

Keywords:
CRISPR/CasProtospacer adjacent motif (PAM)RecognizeSpRY

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are powerful genome editing tools.
  • The utility of CRISPR-Cas systems is often constrained by the availability of specific protospacer adjacent motif (PAM) sequences.
  • SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), shows promise for overcoming PAM limitations in genome editing.

Purpose of the Study:

  • To comprehensively identify and characterize the protospacer adjacent motif (PAM) sequences recognized by the SpRY variant.
  • To evaluate the efficiency and specificity of SpRY binding to various PAM sequences in human cells.
  • To provide essential data for optimizing SpRY-based genome editing strategies in human therapeutics and research.

Main Methods:

  • Utilized the established PAM Definition by Observable Sequence Excision (PAM-DOSE) system.
  • Employed a green fluorescent protein (GFP)-reporter assay for evaluating SpRY activity.
  • Tested a range of potential PAM sequences in human cell lines to determine SpRY recognition.

Main Results:

  • Identified 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' as canonical PAM sequences for SpRY.
  • Determined 5'-NCA-3' and 5'-NTK-3' as potential non-priority PAM sequences.
  • Found that the 5'-NYC-3' PAM sequence is not recommended for SpRY in human cells.

Conclusions:

  • The identified PAMs provide crucial guidance for the effective application of SpRY in human genome editing.
  • This research expands the toolkit for CRISPR-Cas genome engineering by detailing SpRY's PAM compatibility.
  • Findings facilitate more precise and versatile SpRY-mediated gene targeting in human cells.