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

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.

Expanding the scope of bacterial CRISPR activation with PAM-flexible dCas9 variants

“…However, because we desire to express a single dCas9 protein to simultaneously target multiple genes for CRISPRa or CRISPRi, it is important to evaluate the performance of expanded PAM variants for CRISPRi. Previous work in bacterial and eukaryotic systems suggests that expanded PAM variants exhibit impaired CRISPRi function (Legut et al, 2020; Wang et al, 2021). We proceeded to evaluate the PAM-flexible variants dxCas9(3.7), dxCas9-NG, and dSpRY for CRISPRi gene repression.…”

“…Previous work in bacterial and eukaryotic systems suggests that expanded PAM variants exhibit impaired CRISPRi function (Legut et al, 2020;Wang et al, 2021). We proceeded to evaluate the PAM-flexible variants dxCas9(3.7), dxCas9-NG, and dSpRY for CRISPRi gene repression.…”

Improving isoprenol productionviasystematic CRISPRi screening in engineeredEscherichia coli

“…To facilitate the screening, we utilized a previously engineered mevalonate over-production strain E. coli BW25113(F′)::MVA, which harbors a synthetic P L lacO1-controlled mevalonate pathway integrated at the aslB locus in the genome of E. coli BW25113(F′) and can produce mevalonate with a yield of 0.225 g −1 glucose. 37 The P L lacO1-controlled dCas9 was then integrated at the low-expression dkgB locus, 38,39 yielding strain E. coli BW25113(F′)::MVA9 (Fig. 1).…”

“…The fatty acid biosynthesis pathway is one of the major acetyl-CoA consumption pathways and is essential for cell growth, whose repression are beneficial to reserve the acetyl-CoA pool by either supplementation of antibiotic cerulenin or transcriptional repression. 37,42,43 In this study, three essential genes involved in fatty acid biosynthesis initiation were targeted, including the acetyl-CoA carboxyltransferase subunit α gene (accA), and the malonyl-CoA-acyl carrier protein transacylase gene ( fabD), and the β-ketoacyl-[acyl carrier protein] synthase 3 ( fabH) (Fig. 1).…”

“…To circumvent the detrimental effects of CRISPRi on cell fitness, we chose to repress the selected essential genes with shortened spacers and different targeting positions that have been demonstrated effective in tuning the gene repression efficiency. 29,33,37,38,44 For the production test in shake flasks, E. coli BW25113(F′)::MVA9 was co-transformed with pZE-PMD-MK-AphA and pCS-sgRNAs harboring sgRNAs containing 10 bp spacers targeting two different locations within the coding sequence of accA, fabD and fabH (Fig. 3A).…”