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
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.
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.
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