Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks

Abstract

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

Versatile transcription control based on reversible dCas9 binding

“…1C). For both T7 RNAP and Pol II, Clarke and coworkers found that the quantity of free, active Cas9 resulting from displacement varies significantly among different tDNA-targeting sgRNAs, but nearly no displacement occurs with any ntDNA-targeting sgRNA tested (Clarke et al 2018). Again consistent with these results, we found that all four of the ntDNA-targeting sgRNAs we tested almost fully blocked transcription by T7 RNAP.…”

“…1C), analogous to in vivo observations of transcription by Eco RNAP (Qi et al 2013). Clarke et al (2018) investigated the ability of transcribing T7 RNAP and Pol II to displace an sgRNA:WT Cas9 complex following DNA cleavage by Cas9. Testing a broad panel of sgRNAs in vitro, they found that Cas9 generally gets displaced by RNAP when the sgRNA targets the tDNA and does not get displaced when the sgRNA targets the ntDNA.…”

“…We note that the blockage efficiency and the stability of RNAP at the blockade may be affected by factors such as the target sequence and target strand (which determines the orientation of the blockade relative to the approaching RNAP). Clarke et al investigated the ability of transcribing T7 RNAP and Pol II to displace an sgRNA:WT Cas9 complex following DNA cleavage (Clarke et al 2018). Testing a broad panel of sgRNAs in vitro, they found that Cas9 generally gets displaced by both RNAPs when the sgRNA targets the tDNA, but not when it targets the ntDNA.…”

The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies

“…However, it is challenging to disentangle transcription from chromatin factors that vary with transcriptional state. Interestingly, a recent study found that only particular sgRNAs showed higher mutation rates targeting a transcriptionally activated gene compared to its uninduced state [35]••. Cas9:sgRNA complexes that directly base-paired to the DNA strand serving as a template for RNA polymerase, not those binding the complementary strand, showed an increase in mutagenesis rate.…”

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage

“…Genomic inaccessibility limits the number of possible Cas9 recognition sites, [ 53,54 ] preventing a large number of potential off‐target cuts. Similarly, Cas9 competes with other DNA‐binding proteins such as polymerases, [ 55 ] transcription factors, [ 12 ] histones, [ 53 ] and histone chaperones. [ 54,56 ]…”