Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems

Abstract

Introduction: Clustered regularly interspaced short palindromic repeat and its associated protein (CRISPR-Cas)-based technologies generate targeted modifications in host genome by inducing site-specific double-strand breaks (DSBs) that can serve as a substrate for homology-directed repair (HDR) in both in vitro and in vivo models. HDR pathway could enhance incorporation of exogenous DNA templates into the CRISPR-Cas9-mediated DSB site. Owing to low rate of HDR pathway, the efficiency of accurate genome editing is diminished. Enhancing the efficiency of HDR can provide fast, easy, and accurate technologies based on CRISPR-Cas9 technologies. Methods: The current study presents an overview of attempts conducted on the precise genome editing strategies based on small molecules and modified CRISPR-Cas9 systems. Results: In order to increase HDR rate in targeted cells, several logical strategies have been introduced such as generating CRISPR effector chimeric proteins, anti-CRISPR proteins, modified Cas9 with donor template, and using validated synthetic or natural small molecules for either inhibiting non-homologous end joining (NHEJ), stimulating HDR, or synchronizing cell cycle. Recently, high-throughput screening methods have been applied for identification of small molecules which along with the CRISPR system can regulate precise genome editing through HDR. Conclusion: The stimulation of HDR components or inhibiting NHEJ can increase the accuracy of CRISPR-Cas-mediated engineering systems. Generating chimeric programmable endonucleases provide this opportunity to direct DNA template close proximity of CRISPR-Cas-mediated DSB. Small molecules and their derivatives can also proficiently block or activate certain DNA repair pathways and bring up novel perspectives for increasing HDR efficiency, especially in human cells. Further, high throughput screening of small molecule libraries could result in more discoveries of promising chemicals that improve HDR efficiency and CRISPR-Cas9 systems.

Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients

“…ssODN-mediated DNA editing relies on HDR, which is less effective than the NHEJ pathway in mammalian cells. 21 , 37 Therefore, we used an HDR activator (L755507) and an NHEJ inhibitor (M3814) to improve HDR-mediated DNA editing, as previously reported. 37 In addition, Cas9 delivery type has been found to be significant in on-target gene editing efficiency and in avoiding undesirable genomic modifications.…”

“… 21 , 37 Therefore, we used an HDR activator (L755507) and an NHEJ inhibitor (M3814) to improve HDR-mediated DNA editing, as previously reported. 37 In addition, Cas9 delivery type has been found to be significant in on-target gene editing efficiency and in avoiding undesirable genomic modifications. 21 , 31 In this sense, plasmid-based Cas9 overexpression in edited cells has been related to lower knockin efficiencies and to a dramatic increase in the re-cutting of edited sites.…”

CRISPR-Cas9 RAG2 Correction via Coding Sequence Replacement to Preserve Endogenous Gene Regulation and Locus Structure

“…Thus, reduction of the rAAV6 toxicity is required in order for a rAAV6-based method to be implemented as a clinical therapy for the purpose of gene correction. Follow-up studies to test different methods of improving HDR to allow for further reduction of the MOI, including inhibiting the NHEJ repair pathway via molecules such as i53 and/or DNA-PK inhibitors [75][76][77] , are currently underway.…”

Metabolic priming of GD2TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

“…3A). Lower basal respiration (MP: 32 pmol/min (15) versus Control: 180 pmol/min (140), p = 0.02), maximal respiration (MP: 72 pmol/min (41) versus Control: 298 pmol/min (74), p = 0.002), and spare respiratory capacity (MP: 40 pmol/min (27) versus Control: 118 pmol/min (66), p = 0.03) were seen in MP compared to Control TRAC-CAR T cells (Fig. 3A).…”

“…T cells have classically been transduced with γ-retroviral or lentiviral vectors yielding stable but uncontrolled genomic integration of the CAR transgene with a constitutively active promoter, 29,31 which has been implicated in excessive T-cell differentiation. 33 CRISPR/Cas9 is an alternative approach that makes a double-stranded DNA break at a precise genomic locus where the DNA repair pathways can stably integrate the desired CAR transgene. [33][34][35] This genome editing strategy has been used to insert a CAR transgene upstream of the endogenous T-cell receptor alpha constant (TRAC) gene using homologydirected repair (HDR).…”