CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10 12 ). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N 20 ) to 9 nucleotides (N 9 ) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5′-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl − phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular L-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.

With developments in synthetic biology, “engineering biology” has emerged through standardization and platformization based on hierarchical, orthogonal, and modularized biological systems. Genome engineering is necessary to manufacture and design synthetic cells with desired functions by using bioparts obtained from sequence databases. Among various tools, the CRISPR-Cas system is modularly composed of guide RNA and Cas nuclease; therefore, it is convenient for editing the genome freely. Recently, various strategies have been developed to accurately edit the genome at a single nucleotide level. Furthermore, CRISPR-Cas technology has been extended to molecular diagnostics for nucleic acids and detection of pathogens, including disease-causing viruses. Moreover, CRISPR technology, which can precisely control the expression of specific genes in cells, is evolving to find the target of metabolic biotechnology. In this review, we summarize the status of various CRISPR technologies that can be applied to synthetic biology and discuss the development of synthetic biology combined with CRISPR technology in microbiology.