An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing.

Nannan Duan,Shuqing Tang,Miaojin Zhou,Desheng Liang,Lingqian Wu,Baitao Zeng,Qian Hu,Zhiqing Hu

doi:10.3390/life11111262

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.

Highlights

The Cas12a system was designed by removing the OriP/EBNA1 elements of Cas12a with Orip/EBNA1 (COE). (b) Schematic of genome editing in 293T-Green Fluorescent Protein (GFP). (c) Flow results of the GFP positive rates of cells on 15 and 21 days after transfection
The rate of positive cells did not decrease further during the detection period (Figure 1c). These results demonstrated that the COE system has a higher editing efficiency than Cas12a and Cas9, as this advantage became more obvious with the increase in cultivation time
An episomal clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a gene-editing system was established based on Orip/EBNA1 (COE) and its editing efficiency in HEK-293T and induced pluripotent stem cells (iPSCs) was evaluated

Summary

Introduction

Gene editing techniques, including zinc finger nucleases (ZFNs) [1,2], transcription activator-like effector nucleases (TALENs) [3,4,5], and clustered regularly interspaced short palindromic repeats (CRISPR) [6,7,8], have been harnessed as powerful tools for manipulating the genome in a myriad of organisms. The CRISPR and CRISPR-associated protein (Cas) system has redefined biomedical research due to its simplicity and ease of design. In this system, Cas proteins generate double-stranded breaks (DSBs) in specific sites. DSBs will be repaired by the cell’s endogenous DNA repair machinery through non-homologous end-joining (NHEJ) or homology-directed repair (HDR), resulting in small insertions and deletions (indels) that have specific effects on gene expression [6]

Methods

Results

Conclusion