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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.

Pretreatment with pertussis toxin or microinjection of guanosine-5'-(3-thiotriphosphate) (CTP-7-S) into guard cells in peeled epidermis of Commelina communis 1. promoted stomatal opening under subsaturating white light. Guanosine-5'-(2-thiodiphosphate) (CDP-8-9 and adenosine-5'-(3-thiotriphosphate) (ATP-7-S) did not change stomatal aperture under identical conditions. These results indicate that C proteins may be involved in the regulation of stomatal opening.

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.