Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

“…Currently, many Clostridium studies have designed multiple single-guide RNAs (sgRNAs) and gRNAs in the CRISPR-Cas systems to acquire mutations efficiently, which is limited for the rapid genome engineering in Clostridium research ( 7 ). Unlike the sgRNA selection in CRISPR-Cas9 systems by various computational tools ( 25 ), the identification of gRNAs with high mutation efficiency remains a challenge for CRISPR-Cas12a systems in diverse species ( 31 , 44 , 45 ). Initially, we used the web tool CRISPOR ( 46 ) to select target sites for pre-crRNA, with TTTV as the PAM for both AsCas12a and FnCas12a.…”

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

“…By enhancing the specificity and precision of CRISPR/Cas9 delivery, biomaterials should be engineered to reduce off-target effects. This might include improving the design of the biomaterials or using improved CRISPR/Cas9 variants with increased selectivity ( Feng et al, 2022 ; Iqbal et al, 2023 ). 2.…”

A calibrated cell‐based functional assay to aid classification of MLH1 DNA mismatch repair gene variants

“…The ability to scale up the use of CRISPR gene editing for testing VUS may help overcome these challenges. CRISPR technology has advanced rapidly with new approaches to improve efficiency constantly being developed (Feng et al, 2022;Skarnes et al, 2019). However, until the use of a CRISPR-based assay in cells becomes more practical for clinical laboratories, we recommend a variant-testing strategy that utilizes the previously calibrated CIMRA assay as a first test to identify those variants that clearly disrupt MMR function, as it seems unlikely that variants which severely affect biochemical activity in vitro would display proficient function in the cell (Drost et al, 2010(Drost et al, , 2019González-Acosta et al, 2020).…”