Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Abstract

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope

“…introduction of a compatible exogenous recombination repair system or elevating the expression of host DNA repair proteins). Consistent with our results, a recent study has revealed that attenuating the DNA targeting activity of Cas9 or Cas12a in bacteria boosts the transformation efficiency without compromising gene editing efficiency under certain cases 64 .…”

An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition betweenKlebsiellaspecies

“…Furthermore, we observed that sgRNA, which gave rise to more escapers, also allowed better editing when the lambda Red genes were induced, resulting in a higher editing efficiency. This was recently demonstrated in a study that obtained an increased editing efficiency by attenuating sgRNA 29 . While designing multiple sgRNAs can circumvent this heterogeneity for the purpose of gene deletion, in the case of other edits aimed at a specific genomic location, attenuation could potentially be useful for sgRNAs yielding no colonies, such as sg189 (Figure 1d).…”

“…For sg185, alignment of the targets and flanking PAM sequences to genomic sequences revealed 100% sequence identity between the K. oxytoca and K. michiganensis strains but two mismatches between both K. grimontii strains and sg185 (Figure 3f). Notably, Cas9 has been reported to retain functionality despite mismatches between the sgRNA guide and genomic target 29 , and these mismatches were not anticipated to preclude successful targeting in K. grimontii . A non-targeting guide RNA encoding pSG-spe was included as a control for transformation efficiency.…”

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V–F CRISPR/Cas Nuclease AsCas12f1

“…This was possibly due to its weak dsDNA cleavage ability, thereby enabling cells to have sufficient time repairing DSBs with other uncleaved chromosomal copies. 39 In addition, due to the small DNA size of AsCas12f1, which is nearly 1/3 of the size of two most SpCas9 and FnCpf1, 24 the transformation efficiency of AsCas12f1-based recombinant plasmids (without sgRNAs) is significantly higher than those of SpCas9 and FnCpf1-based ones. Meanwhile, we showed that in the absence of specific sgRNAs, AsCas12f1 induced significantly lower toxicity to S. coelicolor M145 than SpCas9 (Figure 1B), which may be explained by less nonspecific DNA-binding mainly due to the much lower distribution rate of 5′-NTTR PAMs recognized by AsCas12f1 (an average of 10.9 PAMs per gene) compared with 3′-NGG of SpCas9 (an average of 220.0 PAMs per gene) (Figure S2).…”

“…In comparison with SpCas9/sgRNAs and FnCpf1/crRNAs-based chromosomal cleavage that resulted in cell death (showing high host toxicity), AsCas12f1/sgRNAs-based DNA cleavage led to the formation of a high amount of exconjugants (exhibiting lower toxicity). This was possibly due to its weak dsDNA cleavage ability, thereby enabling cells to have sufficient time repairing DSBs with other uncleaved chromosomal copies . In addition, due to the small DNA size of AsCas12f1, which is nearly 1/3 of the size of two most SpCas9 and FnCpf1, the transformation efficiency of AsCas12f1-based recombinant plasmids (without sgRNAs) is significantly higher than those of SpCas9 and FnCpf1-based ones.…”