In silico sgRNA tool design for CRISPR control of quorum sensing in Acinetobacter species

Abstract

CRISPR genome editing utilizes Cas9 nuclease and single guide RNA (sgRNA), which directs the nuclease to a specific site in the genome and makes a double-stranded break (DSB). Design of sgRNA for CRISPR-Cas targeting, and to promote CRISPR adaptation, uses a regulatory mechanism that ensures maximum CRISPR-Cas9 system functions when a bacterial population is at highest risk of phage infection. Acinetobacter baumannii is the most regularly identified gram-negative bacterium infecting patients. Recent reports have demonstrated that the extent of diseases caused by A. baumannii is expanding and, in a few cases, now surpasses the quantity of infections caused by P. aeruginosa. Most Acinetobacter strains possess biofilm-forming ability, which plays a major role in virulence and drug resistance. Biofilm bacteria use quorum sensing, a cell-to-cell communication process, to activate gene expression. Many genes are involved in biofilm formation and the mechanism to disrupt the biofilm network is still not clearly understood. In this study, we performed in silico gene editing to exploit the AbaI gene, responsible for biofilm formation. The study explored different tools available for genome editing to create gene knockouts, selecting the A. baumannii AbaI gene as a target.

Antibiotic Resistance Profiles, Molecular Mechanisms and Innovative Treatment Strategies of Acinetobacter baumannii

“…For this, they used a series of genetic editing tools to create AbaI gene knockouts. The analyzed tools (CHOCHOP, CCTop, E-CRISP, CRISPR Direct, Off-Spotter, Crispr-era) can provide information about the target sequences, specific primers, existing mutations, the location of the target sequence in order to perform the knockout, as well as the necessary sgRNA sequences for performing genomic editing experiments [ 335 ]. The information obtained from the in silico genomic experiments demonstrates the need to improve genetic editing tools.…”

<p>Trends and Development in the Antibiotic-Resistance of <em>Acinetobacter baumannii</em>: A Scientometric Research Study (1991–2019)</p>

“…47 Some studies have proposed using the CRISPR system to combat antibiotic resistance and achieved certain research results. [48][49][50] Now the focus of the research is to solve the limitation of therapy in clinical treatment, then apply it to the clinical widely. The combination of antibiotics and phages is based on the treatment of antibiotic-resistant A. baumannii, while the study of biofilm is based on the control of the transmission of antibiotic-resistant A. baumannii.51 Both of them are to solve the problem of antibiotic-resistance of A. baumannii, but the control of transmission is to prevent the problem at the source, which is more effective to solve the problem.…”

“… 47 Some studies have proposed using the CRISPR system to combat antibiotic resistance and achieved certain research results. 48 – 50 Now the focus of the research is to solve the limitation of therapy in clinical treatment, then apply it to the clinical widely.…”

Recent Advances in Genetic Tools for Acinetobacter baumannii

“…At the core of these approaches remain NGS and subsequent data analysis to identify functional cleavage sites, in combination with genome wide techniques including double-strand break (DSB) capture and protein binding site capture ( Lee et al, 2016 ). Another study ( Karlapudi et al, 2018 ) using A. baumannii strain M2, evaluated the application of web-based tools (CHOPCHOP, CRISPR-ERA, CCTop and E-CRISP) for in silico sgRNA design. Individual web-based tools consider one or multiple relevant factors that includes location of (PAM), sequence alignment scores attributed to GC content and exon position thereby predicting on-target efficacy.…”