Barriers to genome editing with CRISPR in bacteria

Vento, Justin M.; Crook, Nathan; Beisel, Chase L.

doi:10.1007/s10295-019-02195-1

Cited by 92 publications

(102 citation statements)

References 103 publications

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“…When mutations are introduced into the bacterial genome via oligonucleotide-directed mutagenesis, unedited cells are expected to be eliminated by DSBs at unchanged targets by CRISPR/Cas9, and only the edited cells are expected to survive; this is called negative selection. Thus Cold Spring Harbor Laboratory Press on September 8, 2020 -Published by genome.cshlp.org Downloaded from far, various CRISPR-based genome editing technologies have been developed to induce mutations including substitution and indels across diverse bacterial species for basic genetic studies and biotechnological applications (Vento et al 2019).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

2020

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Genome editing has been revolutionized by the CRISPR/Cas9 system. CRISPR/Cas9 is composed of single molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a PAM (protospacer adjacent motif) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR/Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR/Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced 2-to 4-base mutations in galK in E. coli with high editing efficiencies (8186%). However, single point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed 1-or 2-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli. A single point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 3695% of negatively selected E. coli cells on using single base-mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

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Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

2020

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“…Base editing bypasses the general bottlenecks of applying CRISPR-Cas systems in bacteria, which include the toxicity of Cas nucleases and inefficient DNA repairing mechanisms. It also lowers the requirement of transformation efficiency in C. ljungdahlii compared to conventional CRISPR-Cas-based genome editing (10,12,16). We discovered a great capability of our base-editing tool.…”

Section: An Expanded Synthetic Biology Toolkit For Acetogenic Bacteriamentioning

confidence: 93%

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

Xia

Casini

Schulz

et al. 2020

Preprint

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Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-tothymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome.Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient genemanipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a onenucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

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“…The application of CRISPR-Cas gene editing to bacteria has been limited, compared to the rapid rise in use in eukaryotes (reviewed in [13][14][15]. This stems in part from the poor capability of model bacteria to repair CRISPR-generated DSBs by NHEJ.…”

Section: Introductionmentioning

confidence: 99%

The application of the CRISPR–Cas9 system in Pseudomonas syringae pv. actinidiae

Zhao

Wojcik

et al. 2020

Journal of Medical Microbiology

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Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world. Aim. We aim to introduce a CRISPR–Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species. Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR–Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa. Results. We have constructed plasmids carrying a CRISPR–Cas9 system based on that of S. pyogenes , which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR–Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site. Conclusion. We have constructed shuttle plasmids carrying a CRISPR–Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.

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Barriers to genome editing with CRISPR in bacteria

Cited by 92 publications

References 103 publications

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

The application of the CRISPR–Cas9 system in Pseudomonas syringae pv. actinidiae

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