CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

Abstract

Genome editing has been revolutionized by the CRISPR/Cas9 system. CRISPR/Cas9 is composed of single molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a PAM (protospacer adjacent motif) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR/Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR/Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced 2-to 4-base mutations in galK in E. coli with high editing efficiencies (8186%). However, single point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed 1-or 2-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli. A single point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 3695% of negatively selected E. coli cells on using single base-mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

“…E. coli DH5α was used as a cloning host for the construction of the sgRNA plasmids. E. coli MG1655 was constructed to harbor the cas9 [ 26 ] or cas9-NG gene in their chromosomes. The construction of an E. coli strain carrying the cas9-NG gene in the chromosome is described below.…”

“…Nevertheless, single-base-level genome editing is rarely achieved, even in microbial cells, which is likely due to mismatch tolerance. We recently used CRISPR/Cas9 and Cpf1 to design a mismatched sequence in the sgRNA target recognition sequence (TRS) in advance, to overcome mismatch tolerance, thereby enabling site-specific single-base editing in a bacterial cell with high efficiency [ 25 , 26 ]. Here, we demonstrate a simple and effective single-base microbial genome editing method that can overcome single-base mismatch tolerance using a 5′-truncated sgRNA/Cas9 complex.…”

CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit forE. coli

“…As a result, in many bacteria, including E. coli, CRISPR-Cas9 has been widely employed as a tool for counter-selection to eliminate non-modified cells from a mixed population for enrichment of gene-modified cells by homology-directed recombination methods such as the lambda red recombination systems 20,21 . It remains very challenging to engineer DNA at a single nucleotide level, even when combined with a powerful counter-selection system such as CRISPR-Cas9, the efficiency of making point mutations using oligonucleotide-directed mutagenesis is very low (< 3% before optimization) 22 .…”

Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference

“…From a bacterial adaptive immune system, CRISPR/Cas technology has been evolved and used for accurate and efficient genome editing [8][9][10][11]. However, there is still a drawback that the PAM sequence should recognized along with the designed DNA target sequence.…”