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

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.

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Related in: MedlinePlus

One-step inactivation of chromosomal gene(s) by CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ).Cas9 and NHEJ-related proteins (Mt-Ku and Mt-LigD) are expressed in host cells, which are then transformed with a single-guide RNA (sgRNA) donor plasmid to generate double-stranded breaks (DSBs) and trigger indel mutations. Mutagenesis is attributed to the RNA-directed Cas9 cleavage system and the error-prone NHEJ repair system. First, site-specific DSB is generated via sgRNA-directed Cas9 cleavage. The DNA ends are recognized and stabilized by the DNA end-binding protein Mt-Ku. Next, the ATP-dependent DNA ligase Mt-LigD is recruited to the DNA ends; the imprecise repair of DSB results in a frameshift mutation. Finally, only the DSB-repaired colonies lacking the Cas9 targeting site survive CRISPR-Cas9 screening. To further engineer the strain, the sgRNA donor plasmid is cured via an inducible sgRNA-mediated “suicide” strategy, and the temperature-sensitive plasmid pCas9 (Ts)-NHEJ by growing the cells at 42 °C.
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f1: One-step inactivation of chromosomal gene(s) by CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ).Cas9 and NHEJ-related proteins (Mt-Ku and Mt-LigD) are expressed in host cells, which are then transformed with a single-guide RNA (sgRNA) donor plasmid to generate double-stranded breaks (DSBs) and trigger indel mutations. Mutagenesis is attributed to the RNA-directed Cas9 cleavage system and the error-prone NHEJ repair system. First, site-specific DSB is generated via sgRNA-directed Cas9 cleavage. The DNA ends are recognized and stabilized by the DNA end-binding protein Mt-Ku. Next, the ATP-dependent DNA ligase Mt-LigD is recruited to the DNA ends; the imprecise repair of DSB results in a frameshift mutation. Finally, only the DSB-repaired colonies lacking the Cas9 targeting site survive CRISPR-Cas9 screening. To further engineer the strain, the sgRNA donor plasmid is cured via an inducible sgRNA-mediated “suicide” strategy, and the temperature-sensitive plasmid pCas9 (Ts)-NHEJ by growing the cells at 42 °C.

Mentions: The scheme of the CA-NHEJ strategy for one-step gene(s) inactivation is presented in Fig. 1. First, Cas9 from Streptococcus pyogenes SF370 and the conserved prokaryotic NHEJ proteins from Mycobacterium tuberculosis H37Rv were heterologously expressed in E. coli. The sgRNA expression plasmid, containing the information for Cas9 targeting via Watson–Crick base pairing, was then electrotransformed into the host, resulting in the site-specific generation of DSBs and deletion mutations. Briefly, the sgRNA-Cas9 complex binds and cleaves the target DNA strands that generate DSBs at desired genomic loci, which is lethal to wild-type E. coli24. The imported NHEJ pathway locates and repairs the DSB, such that the cells survive in CRISPR-Cas9 cleavage but carry mutations in the targeted genomic locus. Finally, the helper plasmid is cured through a “suicide” strategy in which an inducible sgRNA targeting the plasmid is expressed and Cas9-mediated cleavage occurs. The overall procedure of gene inactivation by the CA-NHEJ system is remarkably simple—as its only requirements are the construction of a specific sgRNA and single-step electroporation—and thus accelerates the process of genome engineering.


A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome
One-step inactivation of chromosomal gene(s) by CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ).Cas9 and NHEJ-related proteins (Mt-Ku and Mt-LigD) are expressed in host cells, which are then transformed with a single-guide RNA (sgRNA) donor plasmid to generate double-stranded breaks (DSBs) and trigger indel mutations. Mutagenesis is attributed to the RNA-directed Cas9 cleavage system and the error-prone NHEJ repair system. First, site-specific DSB is generated via sgRNA-directed Cas9 cleavage. The DNA ends are recognized and stabilized by the DNA end-binding protein Mt-Ku. Next, the ATP-dependent DNA ligase Mt-LigD is recruited to the DNA ends; the imprecise repair of DSB results in a frameshift mutation. Finally, only the DSB-repaired colonies lacking the Cas9 targeting site survive CRISPR-Cas9 screening. To further engineer the strain, the sgRNA donor plasmid is cured via an inducible sgRNA-mediated “suicide” strategy, and the temperature-sensitive plasmid pCas9 (Ts)-NHEJ by growing the cells at 42 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121644&req=5

f1: One-step inactivation of chromosomal gene(s) by CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ).Cas9 and NHEJ-related proteins (Mt-Ku and Mt-LigD) are expressed in host cells, which are then transformed with a single-guide RNA (sgRNA) donor plasmid to generate double-stranded breaks (DSBs) and trigger indel mutations. Mutagenesis is attributed to the RNA-directed Cas9 cleavage system and the error-prone NHEJ repair system. First, site-specific DSB is generated via sgRNA-directed Cas9 cleavage. The DNA ends are recognized and stabilized by the DNA end-binding protein Mt-Ku. Next, the ATP-dependent DNA ligase Mt-LigD is recruited to the DNA ends; the imprecise repair of DSB results in a frameshift mutation. Finally, only the DSB-repaired colonies lacking the Cas9 targeting site survive CRISPR-Cas9 screening. To further engineer the strain, the sgRNA donor plasmid is cured via an inducible sgRNA-mediated “suicide” strategy, and the temperature-sensitive plasmid pCas9 (Ts)-NHEJ by growing the cells at 42 °C.
Mentions: The scheme of the CA-NHEJ strategy for one-step gene(s) inactivation is presented in Fig. 1. First, Cas9 from Streptococcus pyogenes SF370 and the conserved prokaryotic NHEJ proteins from Mycobacterium tuberculosis H37Rv were heterologously expressed in E. coli. The sgRNA expression plasmid, containing the information for Cas9 targeting via Watson–Crick base pairing, was then electrotransformed into the host, resulting in the site-specific generation of DSBs and deletion mutations. Briefly, the sgRNA-Cas9 complex binds and cleaves the target DNA strands that generate DSBs at desired genomic loci, which is lethal to wild-type E. coli24. The imported NHEJ pathway locates and repairs the DSB, such that the cells survive in CRISPR-Cas9 cleavage but carry mutations in the targeted genomic locus. Finally, the helper plasmid is cured through a “suicide” strategy in which an inducible sgRNA targeting the plasmid is expressed and Cas9-mediated cleavage occurs. The overall procedure of gene inactivation by the CA-NHEJ system is remarkably simple—as its only requirements are the construction of a specific sgRNA and single-step electroporation—and thus accelerates the process of genome engineering.

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