Advances in Accurate Microbial Genome-Editing CRISPR Technologies

Abstract

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 ...

Recent Advances in CRISPR-Cas Technologies for Synthetic Biology

“…Accurate genome editing with Cas dsDNA nuclease and target-mismatched and truncated guide RNAs has also been developed (Lee & Lee, 2021 ) (Fig. 2 ).…”

“…CRISPR-Cas technology has been applied as a genome editing tool for various organisms, including prokaryotes and human cells (Jinek et al, 2012 ). Currently, highly specific and trans- cleaving nucleolytic activities of CRISPR-Cas are used for accurate genome editing (Lee & Lee, 2021 ) and diagnosis (Kaminski et al, 2021 ), respectively. Deactivated Cas protein is used not only to regulate the transcription of target genes but also to reveal the function of genes related to specific phenotypes through CRISPR screening.…”

“…In microbiology, CRISPR technology has advanced in the following directions. CRISPR genome editing tools have been developed in various microorganisms (Lee & Lee, 2021 ). The function of recognizing specific sequences in CRISPR-Cas has been expanded and applied to molecular diagnosis (Kim et al, 2021 ).…”

Antimicrobial peptides provide wider coverage for targeting drug‐resistant bacterial pathogens

“…[ 79,109,110 ] Interaction of peptides with the cytoplasmic membrane can facilitate insertion into and translocation across the membrane (with potential intracellular target interaction or inhibition) or disruption of the membrane and lysis of the bacterial cell. [ 78,79,111 ]…”

A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

“…These multiple cloning steps present a bottleneck for the rapid deployment of CRISPR-Cas9 genome engineering. This has motivated efforts to increase the rapidity with which genomes can be edited by using strains with Cas9 expressed from an inducible chromosomal locus ( 10 ), plasmids optimized for rapid fragment exchange ( 16 ), and introduction of repair templates by cotransformation with PCR products ( 10 ) or by recombineering using donor oligonucleotides ( 17 ).…”