A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette

Abstract

As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

Keywords: CRISPR/Cas9; antibiotic resistance cassette; genome editing; protospacer adjacent motif; sequence-independent.

FIGURE 1

Efficient genome editing by employing both an antibiotic resistance cassette (ARC) and the CRISPR/Cas9 system. (A) Schematic chart illustrating the two-step strategy for editing non-essential genes (or any DNA sequences) in E. coli. Firstly, an ARC was inserted into the genome near to the target site through homology-directed repair (HDR). Secondly, the Cas9 complex targeting the ARC sequence cleaved the ARC, and meanwhile the ARC was replaced with the edited sequence through the second step of HDR. The target sequences could be mutated, deleted or inserted with foreign DNA sequences, and the λ-Red recombination system remarkably increased the HDR efficiency in both steps. (B) Verification of the edited mutants by both colony PCR (left) and Sanger DNA sequencing (right). Correct sizes are indicated at the end of arrows, and the positive rates are labeled. The PCR products that formed positive clones were then randomly selected for Sanger DNA sequencing analysis. Detailed sequences can be found in Supplementary Figure S3, and the inserted amilGFP sequence contained its own promoter. M, GeneRuler 1-kb DNA ladder (ThermoFisher Scientific).

FIGURE 2

Optimization of the CRISPR/Cas9 system. (A) Sequence alignment between RepA101 expressed from pCas and pCasM. Alanine is mutated to valine at position 56. (B) Plasmid map for pCasM, which was similar to pCas (Jiang et al., 2015) but contained a RepA mutant (RepA_A56V, refer to A). (C) Comparison of the plasmid curing rates between pCasM and pCas in E. coli MG1655 in the absence of antibiotics. To measure the growth rates, bacteria were grown in medium containing kanamycin.

FIGURE 3

Phenotypic analyses of the mutants. (A) Serially diluted mutants were plated on LB containing IPTG and X-gal. In the TAA mutant, the initial codon (ATG) of lacZ was mutated to a stop codon (TAA), resulting in failure to express β-galactosidase. Consequently, the cells failed to degrade X-gal and remained white on the plate. His, N-terminal His-tagged mutant of lacZ. amilGFP, mutant with amilGFP fused to the 5’-end of the lacZ gene. (B) Western blot analysis to confirm the expression of the N-terminal His-tagged LacZ. About 30 μg of cell lysate and 1 μg of purified recombinant His-tagged LacZ were used for western blot analysis, and the same amounts of protein were used in separate SDS-PAGE, which was stained with Coomassie blue and used as a loading control. (C) Fluorescence microscopy detection of the expression of the AmilGFP-LacZ fusion protein.

FIGURE 4

Modification of essential genes using a modified two-step strategy. (A) Schematic chart illustrating the modified two-step strategy for editing an essential gene with or without an independent promoter. The whole procedure was similar to the method described above (Figure 1) except that a ribosome binding site sequence (BBa_J61101 in this study) was linked to the ARC. To edit the 5′-end or the internal sequence of an essential gene, the ARC-ribosome binding site should be inserted in front of the CDS to ensure efficient expression of the gene. (B) Modification of the frr gene (b0172), an essential gene in E. coli. The 6 × His-tag-encoding DNA sequence was successfully inserted after the initial codon of frr, which was verified by Sanger DNA sequencing. Sequence alignment was performed using the SnapGene software (GSL Biotech).

FIGURE 5

An overview of successive genome editing with the new psgRNA and pCasM two-plasmid system.