Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in <i>E. coli</i> through Transient Inhibition of Host RecA Activity

Moreb, Eirik A.; Hoover, Benjamin; Yaseen, Adam; Valyasevi, Nisakorn; Roecker, Zoe; Menacho‐Melgar, Romel; Lynch, Michael

doi:10.1021/acssynbio.7b00174

Cited by 47 publications

(84 citation statements)

References 58 publications

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“…Meanwhile, as expected, knockout of recA significantly increased the lethality of DSBs in the bacterial chromosome as induced by the CRISPR/Cas9 system (Figure 3a), consistent with the conclusions of previous reports(18, 23). Even though it has been suggested that genome editing can benefit from the blocking of recA expression (18, 39), we argue that via the rational selection of sgRNAs (Figure 3b, Table S9) the FPR of genome editing can be significantly reduced without the tradeoff of genome instability derived from blocking the inherent DNA repairing pathways in bacteria.…”

Section: Resultsmentioning

confidence: 64%

Improved sgRNA design in bacteria via genome-wide activity profiling

Guo

Wang

Guan

et al. 2018

Preprint

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CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its 2 0 success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). 1Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we 2 2 systematically profiled sgRNA activity by co-expressing a genome-scale library dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables 2 5 selecting highly active sgRNAs for any locus across the genome in this model organism. We 2 6 also identified 'resistant' genomic loci with respect to CRISPR/Cas9 activity, notwithstanding 2 7 the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity 2 8 prediction models that were trained on mammalian cell datasets were inadequate when coping 2 9with our results, highlighting the key limitations and biases of previous models. We hence 3 0 developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to 3 1 facilitate the design of CRISPR/Cas9-based genome engineering or screenings in bacteria. We 3 2 also isolated the important sgRNA features that contribute to DNA cleavage and characterized 3 3

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Section: Resultsmentioning

confidence: 64%

Improved sgRNA design in bacteria via genome-wide activity profiling

Guo

Wang

Guan

et al. 2018

Preprint

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“…The genes deoB and thyA and corresponding mutants deoB p.309Stop and thyA 755-762del were cloned into a modified pTRC plasmid in which the laqI and promoter region were replaced by the tetR repressor gene and promotor region derived from plasmid Plasmid pEM-Cas9HF1-recA56 (addgene Addgene plasmid # 89962 (Moreb et al, 2017) M9 minimal media + folAmix, then washed with fresh M9 media without folAmix and plated at different concentration of folAmix. Growth rate values are the mean ± SD of at least 3 biological replicates.…”

Section: Effect Of Deob and Thya Expressionmentioning

confidence: 99%

Evolutionary dynamics determines adaptation to inactivation of an essential gene

Rodrigues

Shakhnovich

2019

Preprint

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Genetic inactivation of essential genes creates an evolutionary scenario distinct from escape from drug inhibition, but the mechanisms of microbe adaptations in such cases remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by serial dilutions. The partial reversal G27->C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low supplement folAmix concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss of function mutations in two genes, thyA and deoB, ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation to low folAmix converge to highly accessible yet suboptimal fitness peak.

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“…Additionally, numerous studies have demonstrated the Cas9 activity is not only dependent on a specific gRNA sequence but also on the genomic context (host organism, cell type, etc.). 16,17 The same gRNAs can have highly variable activity between animal models, cell lines, and even strains of E. coli . [16][17][18] In this study, we confirm that "non-target" sites, representing a significant level of context dependent variability, competitively bind to Cas9/gRNA complexes, and can significantly impact on-target activity of Cas9/gRNA complexes in vivo .…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

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CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA.The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

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Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity

Cited by 47 publications

References 58 publications

Improved sgRNA design in bacteria via genome-wide activity profiling

Improved sgRNA design in bacteria via genome-wide activity profiling

Evolutionary dynamics determines adaptation to inactivation of an essential gene

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

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