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A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

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ABSTRACT

Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

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Efficiency and mutation rates of the hetereologous non-homologous end-joining (NHEJ) pathway in E. coli.(a) Efficiency of the NHEJ system in re-circularizing the in vitro HindIII- or SmaI-digested pUC19 plasmid and the in vivo CRISPR-Cas9 cleaved pUC-lacZ plasmid. The error bars represent standard deviations from three replicate experiments. The results are expressed as colony-forming units (CFU) per μg of plasmid DNA. mku and ligd, derived from M. tuberculosis H37Rv and involved in the NHEJ pathway, are hetereologously expressed in E. coli using a strong constitutive PJ23119 promoter. (b) The mutation rates of the NHEJ system with different artificially created DSBs. The mutation rates are statistically determined based on the proportion of white colonies on the X-gal plate. The error bars represent standard deviations from three replicate experiments.
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f2: Efficiency and mutation rates of the hetereologous non-homologous end-joining (NHEJ) pathway in E. coli.(a) Efficiency of the NHEJ system in re-circularizing the in vitro HindIII- or SmaI-digested pUC19 plasmid and the in vivo CRISPR-Cas9 cleaved pUC-lacZ plasmid. The error bars represent standard deviations from three replicate experiments. The results are expressed as colony-forming units (CFU) per μg of plasmid DNA. mku and ligd, derived from M. tuberculosis H37Rv and involved in the NHEJ pathway, are hetereologously expressed in E. coli using a strong constitutive PJ23119 promoter. (b) The mutation rates of the NHEJ system with different artificially created DSBs. The mutation rates are statistically determined based on the proportion of white colonies on the X-gal plate. The error bars represent standard deviations from three replicate experiments.

Mentions: As proof of concept, a plasmid-based gene inactivation experiment was carried out using CA-NHEJ. In the first step, the sgRNA targeting the lacZ gene on plasmid pUC-lacZ was used together with the CRISPR-Cas9 system to investigate the DSB generation. Plasmid pUC-lacZ was electrotransformed into E. coli DH5α (ΔlacZ) containing the complete CRISPR-Cas9 system targeting the alpha-fragment of lacZ in parallel with plasmid pUC19, which served as the negative control that without sgRNA target site. The result revealed 1.3 × 107 fold decrease of transformation efficiency in pUC-lacZ than that of pUC19, which demonstrated the high efficiency of the CRISPR-Cas9 system and the weak capability of endogenous DSB repair in wild-type E. coli (Fig. 2a). The utility of the NHEJ system was then demonstrated by re-circularization of the restriction endonuclease-linearized plasmid25. A 5′-overhang or blunt end of the DSB in the alpha-fragment of lacZ of plasmid pUC19 was generated by HindIII or SmaI digestion, respectively. Transformants harboring the re-circularized plasmid were easily identified by blue-white screening (see Supplementary Fig. 1). The amount of transformant colonies increased by several dozens of times than that of control, indicating efficient DNA repair of the hetereologous NHEJ mechanism in E. coli (Fig. 2a). Blunt end-joining was slightly more efficient than sticky end-joining, which probably reflected the terminal sequence specificity of the various DSBs. Statistical analysis of the proportion of white colonies demonstrated a low fidelity of the heterologous NHEJ DNA repair system in E. coli (Fig. 2b and Supplementary Fig. 1). Moreover, DSB with sticky end (generated by HindIII digestion) was repaired with slightly greater accuracy than that with blunt end (generated by SmaI digestion) (55.3 ± 4.4% vs. 43.0 ± 4.0%) (Fig. 2b).


A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome
Efficiency and mutation rates of the hetereologous non-homologous end-joining (NHEJ) pathway in E. coli.(a) Efficiency of the NHEJ system in re-circularizing the in vitro HindIII- or SmaI-digested pUC19 plasmid and the in vivo CRISPR-Cas9 cleaved pUC-lacZ plasmid. The error bars represent standard deviations from three replicate experiments. The results are expressed as colony-forming units (CFU) per μg of plasmid DNA. mku and ligd, derived from M. tuberculosis H37Rv and involved in the NHEJ pathway, are hetereologously expressed in E. coli using a strong constitutive PJ23119 promoter. (b) The mutation rates of the NHEJ system with different artificially created DSBs. The mutation rates are statistically determined based on the proportion of white colonies on the X-gal plate. The error bars represent standard deviations from three replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5121644&req=5

f2: Efficiency and mutation rates of the hetereologous non-homologous end-joining (NHEJ) pathway in E. coli.(a) Efficiency of the NHEJ system in re-circularizing the in vitro HindIII- or SmaI-digested pUC19 plasmid and the in vivo CRISPR-Cas9 cleaved pUC-lacZ plasmid. The error bars represent standard deviations from three replicate experiments. The results are expressed as colony-forming units (CFU) per μg of plasmid DNA. mku and ligd, derived from M. tuberculosis H37Rv and involved in the NHEJ pathway, are hetereologously expressed in E. coli using a strong constitutive PJ23119 promoter. (b) The mutation rates of the NHEJ system with different artificially created DSBs. The mutation rates are statistically determined based on the proportion of white colonies on the X-gal plate. The error bars represent standard deviations from three replicate experiments.
Mentions: As proof of concept, a plasmid-based gene inactivation experiment was carried out using CA-NHEJ. In the first step, the sgRNA targeting the lacZ gene on plasmid pUC-lacZ was used together with the CRISPR-Cas9 system to investigate the DSB generation. Plasmid pUC-lacZ was electrotransformed into E. coli DH5α (ΔlacZ) containing the complete CRISPR-Cas9 system targeting the alpha-fragment of lacZ in parallel with plasmid pUC19, which served as the negative control that without sgRNA target site. The result revealed 1.3 × 107 fold decrease of transformation efficiency in pUC-lacZ than that of pUC19, which demonstrated the high efficiency of the CRISPR-Cas9 system and the weak capability of endogenous DSB repair in wild-type E. coli (Fig. 2a). The utility of the NHEJ system was then demonstrated by re-circularization of the restriction endonuclease-linearized plasmid25. A 5′-overhang or blunt end of the DSB in the alpha-fragment of lacZ of plasmid pUC19 was generated by HindIII or SmaI digestion, respectively. Transformants harboring the re-circularized plasmid were easily identified by blue-white screening (see Supplementary Fig. 1). The amount of transformant colonies increased by several dozens of times than that of control, indicating efficient DNA repair of the hetereologous NHEJ mechanism in E. coli (Fig. 2a). Blunt end-joining was slightly more efficient than sticky end-joining, which probably reflected the terminal sequence specificity of the various DSBs. Statistical analysis of the proportion of white colonies demonstrated a low fidelity of the heterologous NHEJ DNA repair system in E. coli (Fig. 2b and Supplementary Fig. 1). Moreover, DSB with sticky end (generated by HindIII digestion) was repaired with slightly greater accuracy than that with blunt end (generated by SmaI digestion) (55.3 ± 4.4% vs. 43.0 ± 4.0%) (Fig. 2b).

View Article: PubMed Central - PubMed

ABSTRACT

Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

No MeSH data available.


Related in: MedlinePlus