Abstract
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system, known as the prokaryotic adaptive immune system, is present in bacteria and archaea [1,2]
We introduced the applications of CRISPR/Cas9 and CRISPR interference (CRISPRi) systems in various bacterial cells for gene deletion, insertion, replacement, and regulation of gene expression
In addition to the rational design, the engineered targets can be obtained through screening of the synthetic single-guide RNA (sgRNA) library, which represents the whole set of bacterial genes
Summary
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system, known as the prokaryotic adaptive immune system, is present in bacteria and archaea [1,2]. The binding of dCas directed by gRNA to the specific genomic locus can efficiently inhibit the progress of RNA polymerase (RNAP) to the downstream gene This simple interference system known as CRISPRi enables the regulation of expression of target genes transiently or constitutively. DCas has been fused with the myc-associated factor X (MAX)-interacting proteins 1 (MXI1), a transcriptional repressor domain interacting with the histone deacetylase Sin homolog in yeast, the Krüppel-associated box (KRAB) domain of Kox, a protein promoting heterochromatin formation, the chromo shadow (CS) domain of HP1α, a protein related in H3K9me3-dependent gene silencing, and the hairy-related basic helix-loop-helix repressor proteins (WRPW) domain of Hes1 [26,27,28,29,30] These modified dCas9s demonstrated more efficient and robust gene inactivation than the simple interference system. CRISPRi, Resveratrol production, Malonyl-CoA pathway (fabD, fabH, fabB, fabF, fabI) (6-fold)
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