CRISPR technologies for bacterial systems: Current achievements and future directions

Choi, Kyeong Rok; Lee, Sang Yup

doi:10.1016/j.biotechadv.2016.08.002

Cited by 130 publications

(89 citation statements)

References 246 publications

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“…Surprisingly, we found that only the K. hansenii clade and K. medellinensis NBRC 3288 strain harbors a CRISPR‐Cas system, in particular, of the Type I‐E, as predicted by the presence of cas3 signature gene and sequence similarity to E. coli K12 proteins (Figure c; Choi & Lee, ; Makarova, Zhang, & Koonin, ). These genomes contain one loci of the cas gene cluster consisting of 4‐20 palindromic repeats of 29 bp, separated by 32‐bp spacers (Supporting Information Table S2).…”

Section: Resultsmentioning

confidence: 85%

Comparative genomics of the Komagataeibacter strains—Efficient bionanocellulose producers

Ryngajłło

Kubiak

Jędrzejczak-Krzepkowska

et al. 2018

MicrobiologyOpen

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Komagataeibacter species are well‐recognized bionanocellulose ( BNC ) producers. This bacterial genus, formerly assigned to Gluconacetobacter , is known for its phenotypic diversity manifested by strain‐dependent carbon source preference, BNC production rate, pellicle structure, and strain stability. Here, we performed a comparative study of nineteen Komagataeibacter genomes, three of which were newly contributed in this work. We defined the core genome of the genus, clarified phylogenetic relationships among strains, and provided genetic evidence for the distinction between the two major clades, the K . xylinus and the K. hansenii . We found genomic traits, which likely contribute to the phenotypic diversity between the Komagataeibacter strains. These features include genome flexibility, carbohydrate uptake and regulation of its metabolism, exopolysaccharides synthesis, and the c‐di‐ GMP signaling network. In addition, this work provides a comprehensive functional annotation of carbohydrate metabolism pathways, such as those related to glucose, glycerol, acetan, levan, and cellulose. Findings of this multi‐genomic study expand understanding of the genetic variation within the Komagataeibacter genus and facilitate exploiting of its full potential for bionanocellulose production at the industrial scale.

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Section: Resultsmentioning

confidence: 85%

Comparative genomics of the Komagataeibacter strains—Efficient bionanocellulose producers

Ryngajłło

Kubiak

Jędrzejczak-Krzepkowska

et al. 2018

MicrobiologyOpen

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“…CRISPR/Cas‐mediated bacterial genome editing relies on counter‐selection by lethal double‐stranded DNA break and low‐efficiency homologous recombination. Multiplex bacterial genome editing is still a great challenge due to the low cell survival rate and the need for multiple large homologous arms (Choi & Lee, ). Recently, novel CRISPR/Cas‐guided cytidine base editing technology has been established in eukaryotic cells, enabling C:G to T:A base pair transition in a guide RNA (gRNA)‐dependent manner without introducing double‐stranded DNA break or a donor template (Komor, Kim, Packer, Zuris, & Liu, ; Nishida et al, ).…”

Section: Introductionmentioning

confidence: 99%

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Wang

Liu

et al. 2019

Biotech & Bioengineering

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CRISPR/Cas9‐guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double‐stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome‐targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome‐targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9‐fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5‐bp editing window to 7‐bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user‐friendly online tool named gBIG was provided for designing guide RNAs for base editing‐mediated inactivation of given genes in any given sequenced genome (http://www.ibiodesign.net/gBIG).

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“…Subsequently, a number of other groups, using modified versions of the protocol proposed by Jiang et al demonstrated the broad applicability and flexibility of the technology in several bacterial species. Thanks to these discoveries, the CRISPR/Cas methodology became the most effective strategy to create gene knock-outs and knock-ins in prokaryotes (for a recent review see Choi and Lee [14]). Two are the major breakthroughs of CRISPR/Cas-technology.…”

Section: Discussionmentioning

confidence: 99%

“…With this strategy, Jiang and co-workers reported mutation efficiencies as high as 65%. Subsequently, other authors fine-tuned the Jiang et al protocol by adding innovative solutions and expanding its application for extensive gene deletions and replacements [14–17]. Thanks to the contribution of all these authors, CRISPR/Cas9 coupled to recombineering is becoming the most effective approach for genome editing in bacteria and in particular in E. coli .…”

Section: Introductionmentioning

confidence: 99%

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

et al. 2017

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BackgroundThe exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.ResultsWe first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions.ConclusionsThis work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users.

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CRISPR technologies for bacterial systems: Current achievements and future directions

Cited by 130 publications

References 246 publications

Comparative genomics of the Komagataeibacter strains—Efficient bionanocellulose producers

Comparative genomics of the Komagataeibacter strains—Efficient bionanocellulose producers

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

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