Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit

Mougiakos, Ioannis; Bosma, Elleke Fenna; Vos, Willem M. de; Kranenburg, Richard van; Oost, John van der

doi:10.1016/j.tibtech.2016.02.004

Cited by 125 publications

(112 citation statements)

References 62 publications

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“…Finally, Hjc, unrelated to the RuvABC complex in bacteria (32), is involved in the resolution of the Holliday junction (31). Thus, it is tempting to speculate that coexpression of archaeal HDR machinery along with Cas9 might overcome some of the obstacles that have been reported in recent bacterial work (7)(8)(9).…”

Section: Discussionmentioning

confidence: 98%

“…Appropriately designed repair templates allow recovery of strains with precise insertions and deletions, allowing unprecedented ability to manipulate the genomes of these diploid (or polyploid) organisms (6). Although Cas9-mediated genome editing has been successfully and broadly implemented in eukaryotes (3), similar progress has not been achieved in prokaryotes, with Cas9-mediated genome editing having been demonstrated in only 10 bacterial genera (7)(8)(9)(10); to our knowledge, it has not been applied in archaea.…”

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confidence: 99%

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Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

133

112

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Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJdependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homologydirected repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.he CRISPR (clustered regularly interspaced palindromic repeats) array and associated cas genes are widespread in microbial genomes (1), where they confer acquired immunity to phage and foreign DNA elements (2). The type IIA system from Streptococcus pyogenes is especially well characterized and has been widely applied as a remarkably effective genome-editing tool (3). During genome editing, heterologous expression of the RNA-guided DNA endonuclease Cas9 and a chimeric singleguide (sg) RNA, comprised of a 20-bp spacer that targets the chromosome and an 80-bp scaffold that binds Cas9, leads to a lethal double-strand break (DSB) at all target sites within the genome that are flanked by a 3′ NGG protospacer adjacent motif (PAM) (4) (Fig. S1). In eukaryotes, the nonhomologous endjoining (NHEJ) repair pathway can mend the DSB by generating simple insertions or deletions at the sgRNA target site, thus preventing additional rounds of Cas9-mediated cleavage (3, 5). Alternatively, the native homology-dependent repair (HDR) pathway can repair the fatal DSB, so long as a repair template that modifies or removes the sgRNA target site is provided, again preventing additional rounds of Cas9-mediated cleavage (3, 5) (Fig. S1). Appropriately ...

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Section: Discussionmentioning

confidence: 98%

mentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

133

112

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“…33−35 spCas9 was employed to introduce DSDBs in prokaryotic genomes. These breaks modestly induced the recombination of a provided rescuing/editing template into the targeted chromosome, resulting in genetically modified cells.…”

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confidence: 99%

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

et al. 2017

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Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45–55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

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“…Importantly, they also showed that extremely high levels of CTD-deleted p53 transiently overexpressed under the strong human cytomegalovirus promoter may mask the detrimental effect of the CTD absence, emphasizing the need for more-physiological experimental conditions. Of note, gene-editing technologies such as transcription activator-like effector nucleases (TALENS) and clustered regularly-interspaced short palindromic repeats (CRISPR) [49,50] should provide an excellent alternative to experiments based on transient expression of genes. Taken together, the abovementioned findings are in agreement with discoveries made more than a decade ago by Espinosa and Emerson who originally pointed out the vital contributions of the CTD to the process of p53-dependent transactivation on chromatin DNA [38].…”

Section: The Roles Of the P53 Ctd In Dna Bindingmentioning

confidence: 99%

The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein

Laptenko

Tong

Manfredi

et al. 2016

Trends in Biochemical Sciences

100

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The p53 tumor suppressor is a transcription factor (TF) that exerts antitumor functions through its ability to regulate the expression of multiple genes. Within the p53 protein resides a relatively short unstructured C-terminal domain (CTD) that remarkably participates in virtually every aspect of p53 performance as a TF. Because these aspects are often interdependent and it is not always possible to dissect them experimentally, there has been a great deal of controversy about the CTD. In this review we evaluate the significance and key features of this interesting region of p53 and its impact on the many aspects of p53 function in light of previous and more recent findings.

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Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit

Cited by 125 publications

References 62 publications

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein

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