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. 2015 Apr;81(7):2506-14.
doi: 10.1128/AEM.04023-14. Epub 2015 Jan 30.

Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system

Yu Jiang  1 , Biao Chen  1 , Chunlan Duan  2 , Bingbing Sun  1 , Junjie Yang  1 , Sheng Yang  3
Affiliations

Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system

Yu Jiang et al. Appl Environ Microbiol. 2015 Apr.

Erratum in

Abstract

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.

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Figures

FIG 1
FIG 1
Construction of the CRISPR-Cas two-plasmid system. The cas9 gene and the sgRNA directing it to the targeted region were separated in pCas and pTarget series. (a) pCas contains the cas9 gene with a native promoter, an arabinose-inducible sgRNA guiding Cas9 to the pMB1 replicon of pTarget, the λ-Red recombination system to improve the editing efficiency, and the temperature-sensitive replication repA101(Ts) for self-curing. sgRNA is displayed with its secondary structure (51). (b) pTarget was constructed to express the targeting sgRNA, with (pTargetT series) or without (pTargetF series) donor DNA as editing templates. Cas9, Cas9 endonuclease; pJ23119, synthetic promoter (38); N20, 20-bp region complementary to the targeting region (38); araC, arabinose-inducible transcription factor; pKD46K, a form of pKD46 in which the bla gene is replaced with the aadA gene that confers kanamycin resistance (21); pTrc99A-spec, a form of pTrc99A, in which bla was replaced by aadA, which confers spectinomycin resistance.
FIG 2
FIG 2
Effects of cas9, targeting sgRNA, donor DNA, and λ-Red in the CRISPR-Cas two-plasmid system. (A) Diagram of the experimental conditions. (a) cas9 was deficient in pCas; (b and c) targeting sgRNA (b) or donor DNA (c) was deficient in pTargeting series; (d and e) λ-Red with (RED+) (e) or without (RED-) (d) induction. (B) Mutation efficiency. The fraction of spectinomycin-resistant (spec) and kanamycin-resistant (kan) or chloramphenicol-resistant (cm) and kanamycin-resistant (kan) CFU calculated from total CFU was determined under the experimental conditions shown under the histogram and depicted in panel A. Data are means ± standard deviations from three independent experiments.
FIG 3
FIG 3
Detailed diagram of continual genome editing with the two-plasmid system.
See this image and copyright information in PMC

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