An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

Abstract

(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.

Advances in CRISPR/Cas9-based research related to soybean [Glycine max (Linn.) Merr] molecular breeding

“…used CRISPR/Cas12a to achieve editing in the soybean FAD2 gene for the first time ( Jiang et al., 2017 ). In addition, large chromosomal segments of the target genome were deleted by Duan et al. (2021) using CRISPR/Cas12a, with an editing efficiency of 91.7% ( Duan et al., 2021 ).…”

“…In addition, large chromosomal segments of the target genome were deleted by Duan et al. (2021) using CRISPR/Cas12a, with an editing efficiency of 91.7% ( Duan et al., 2021 ). In 2023, Liang et al.…”

“…In 2017, Jiang et al used CRISPR/Cas12a to achieve editing in the soybean FAD2 gene for the first time (Jiang et al, 2017). In addition, large chromosomal segments of the target genome were deleted by Duan et al (2021) using CRISPR/Cas12a, with an editing efficiency of 91.7% (Duan et al, 2021). produced CRISPR/Cas12a-edited soybeans in just 45 days, with transformation and gene editing efficiencies of 30% and 50%, respectively (Liang et al, 2023).…”