Fig. 6: Challenges and future direction of CRISPR gene editing applied in CAR T-cell therapy.

A Adverse events and feasible solutions. Off-target mutations are caused by sgRNA recognizing the off-target locus in the genome and making unnecessary cleavage at the wrong locus. Cas-CLOVER, fuses the catalytically inactive dCas9 with the Clo51 (CLOVER) nuclease ___domain and uses two gRNAs to only cleave when the Clo51 nuclease dimerizes. Another approach “spacer-nick” combines the nCas9 with a pair of PAM-out sgRNAs at a long spacer distance. Hence Cas9n is guided by the spacer-nick sgRNAs to two target sequences on opposite strands and nicks both DNA strands at an optimal distance to preserve efficient HDR while minimizing NHEJ events. Chromosomal aberrations, such as large deletions and translocations, are indeed frequent phenomena. Here shows possible instances of chromosomal loss and translocations. The key factor leading to chromosomal aberrations is the generation of DSBs. BE develops precise single-base substitutions at multiple sites enabling DSB-free gene editing, thereby reducing the risk of gene rearrangements. B Traditional delivery methods include viral vectors and electroporation. Viral vectors mainly include LV, AV, and AAV. CRISPR components delivered by viral vectors in the form of DNA may increase the risk of off-target, and viral vectors also have other disadvantages including immunogenicity, cargo size limitations, and lower transduction efficiency. The delivery of electroporation in CAR T-cells may cause cell damage. Peptide delivery is an emerging delivery system for CRISPR components in CAR T-cells. LNP and VLP have the potential for in vivo delivery, but two basic issues need to be considered, that is, whether the relevant cell types can be targeted and avoid cell damage and immune rejection in vivo. Created with BioRender.com.