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

Jian Wang, Yuxi Teng, Ruihua Zhang, Yifei Wu, Lei Lou, Yusong Zou, Michelle Li, Zhong-Ru Xie and Yajun Yan ()
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Jian Wang: University of Georgia
Yuxi Teng: University of Georgia
Ruihua Zhang: University of Georgia
Yifei Wu: University of Georgia
Lei Lou: University of Georgia
Yusong Zou: University of Georgia
Michelle Li: North Oconee High School
Zhong-Ru Xie: University of Georgia
Yajun Yan: University of Georgia

Nature Communications, 2021, vol. 12, issue 1, 1-10

Abstract: Abstract The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5′-NGG-3′ PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5′-CAT-3′ PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5′-NG-3′ PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5′-NRN-3′ PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.

Date: 2021
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DOI: 10.1038/s41467-021-27290-9

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