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Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor.

Jian Wang1, Yuxi Teng1, Ruihua Zhang1

  • 1School of Chemical, Materials and Biomedical Engineering, College of Engineering, University of Georgia, Athens, GA, 30602, USA.

Nature Communications
|November 26, 2021
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Summary

Researchers engineered a deactivated Cas9 protein (SpdCas9) to target a wider range of DNA sequences by relaxing its PAM requirement. This creates a versatile CRISPR-based gene repressor applicable across organisms.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Microbial Genetics

Background:

  • CRISPR-associated Cas9 proteins are powerful tools for genome engineering.
  • The Streptococcus pyogenes Cas9 (SpCas9) has broad applications but is limited by its strict 5'-NGG-3' Protospacer Adjacent Motif (PAM) requirement.
  • This PAM specificity restricts the targeting range of SpCas9 in prokaryotes and eukaryotes.

Purpose of the Study:

  • To repurpose deactivated SpCas9 (SpdCas9) as a universal gene repressor.
  • To engineer SpCas9 variants with relaxed PAM compatibility, specifically targeting the 5'-CAT-3' PAM found in start codons.
  • To expand the DNA target range for SpCas9-based gene repression.

Main Methods:

  • Generated and screened SpdCas9 variants for relaxed PAM compatibility.
  • Utilized stepwise, structure-guided mutations on PAM-interacting and PAM-proximal residues.
  • Developed a variant, SpdNG-LWQT, with preferential binding to 5'-NRN-3' PAMs.

Main Results:

  • Created a PAM-flexible SpdCas9 variant, SpdNG-LWQT, accommodating a broader PAM range.
  • Demonstrated effective gene repression using SpdNG-LWQT in both Escherichia coli and Saccharomyces cerevisiae.
  • Showcased customizable sgRNA design for the SpdNG-LWQT repressor system.

Conclusions:

  • Validated the feasibility of expanding Cas9 PAM specificity through targeted engineering.
  • Established a universal SpdCas9-based gene repressor with relaxed PAM recognition.
  • This engineered repressor broadens the applicability of CRISPR technology for gene regulation.