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

Kim

2021

IJMS

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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.

Section: Methodsmentioning

confidence: 99%

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

Kim

2021

IJMS

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“…The P1 vir phage was used to transfer mutations to other strains via standard P1 transduction. The P1 lysates of chloramphenicol-resistant E. coli SH148 cells were used to transduce the HK1160 ( araBAD :: dcas9 - NG -KmR) 40 strain to generate the SH150 strain. Target genes identified from the sgRNA library were knocked out using the following method.…”

Section: Methodsmentioning

confidence: 99%

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference

2023

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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.

“…PAM specificity can be indirectly defined through the binding affinity and repression of the target gene using catalytically deactivated Cas9 (dCas9) and dCas9-NG. Previous studies have demonstrated that dCas9 and dCas9-NG are less restricted by PAM requirements, suggesting that target binding and target cleavage are mediated by different mechanisms [ 75 , 76 ].…”

Section: Pam-independent Genome Editingmentioning

confidence: 99%

Advances in Accurate Microbial Genome-Editing CRISPR Technologies

J. Microbiol. Biotechnol.

2021

Self Cite

More than three decades ago, Ishino et al. discovered repeating sequences with symmetry structures in the 3'end of the iap gene of Escherichia coli [1]. Given that sequencing data was far scarcer at that time, the authors could not identify prokaryotic homologous sequences containing the aforementioned repeating sequences, and thus their biological significance remained uncharacterized. Further, Mojica et al. identified similar palindrome sequences in the genomes of 20 species of bacteria and archaea. The researchers addressed the need for studies regarding the universality, phylogeny, and biological significance of the repeating sequences [2]. Ruud Jansen and Francisco Mojica referred to this "interspersed short sequence repeat family" as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which is exclusively found in prokaryotes and not in eukaryotes or viruses [3].More recent studies determined that the spacer DNA sequences in CRISPR are derived from extrachromosomal genetic elements such as bacteriophages and conjugative plasmids, suggesting that CRISPR acts as an adaptive immune system in bacteria [4]. Experiments conducted in 2007 confirmed that CRISPR effectively acts as a bacterial adaptive immune system by correlating the inactivation of the cas gene with phage sensitivity in Streptococcus thermophilus, an industrial strain common in dairy products [5]. In 2010, the CRISPR/Cas mechanism was elucidated. The cells that survived infections from agents containing exogenous DNA such as phages or plasmids integrated the foreign sequences into their own CRISPR locus. These sequences were subsequently processed into crRNAs as a guide for Cas nucleases and ultimately interfered with future invasions from entities carrying the same nucleic acid sequences [6]. In August 2012, Jennifer Doudna, Emmanuelle Charpentier, and their colleagues identified the underlying mechanism of these phenomena, including the formation of a dual-RNA complex that directs the Cas9 nuclease to the target site, after which the tracrRNA:crRNA-guided Cas9 nuclease can cleave double-stranded DNA targets adjacent to the 5'-GG dinucleotide protospacer adjacent motif (PAM) [7].The CRISPR/Cas system consists of a target-recognizing RNA and a DNA-cleaving Cas nuclease, which are functionally independent. Therefore, to modify the target DNA sequences, the CRISPR/Cas must only alter the target-binding sequence in the target-recognizing guide RNA [8]. In the CRISPR/Cas9 system, two separate crRNAs and tracrRNAs are linked with a loop to form a chimeric single guide RNA (sgRNA), thus facilitating genome editing [7]. Among the many known Cas nucleases, single-polypeptide Cas9 nuclease is the most widely used [9]. After the Streptococcus pyogenes-derived Cas9 nuclease was first used [7], various orthogonal Cas9 nucleases derived from Staphylococcus aureus [10], Streptococcus thermophilus [11], Neisseria meningitidis [12], Francisella novicida [13], and Campylobacter jejuni [14] were discovered. In vivo genome editing using CRISPR/ Cas9 ...