Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency.

Xi-Dian Tang,Fei Gao,De-Kun Chen,Ming-Jie Liu,Qin-Lei Fan,Wen-Tao Ma

doi:10.3389/fgene.2019.00551

Abstract

The evolution of organisms has provided a variety of mechanisms to maintain the integrity of its genome, but as damage occurs, DNA damage repair pathways are necessary to resolve errors. Among them, the DNA double-strand break repair pathway is highly conserved in eukaryotes, including mammals. Nonhomologous DNA end joining and homologous directed repair are two major DNA repair pathways that are synergistic or antagonistic. Clustered regularly interspaced short palindromic repeats genome editing techniques based on the nonhomologous DNA end joining repair pathway have been used to generate highly efficient insertions or deletions of variable-sized genes but are error-prone and inaccurate. By combining the homology-directed repair pathway with clustered regularly interspaced short palindromic repeats cleavage, more precise genome editing via insertion or deletion of the desired fragment can be performed. However, homologous directed repair is not efficient and needs further improvement. Here, we describe several ways to improve the efficiency of homologous directed repair by regulating the cell cycle, expressing key proteins involved in homologous recombination and selecting appropriate donor DNA.

Highlights

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-Associated (Cas) SystemsPrecise and efficient genomic modification is essential to biological processes, genetic engineering, and other various areas of study
The main reasons for this are the low efficiency of homologous directed repair (HDR) and the poor availability of exogenous DNA as a repair template, which seriously affect HDR as an accurate method of genome editing (Li et al, 2018b)
Previous research has shown that nonhomologous end joining (NHEJ) is more errorprone when repairing double-strand break (DSB), but recent studies have demonstrated that NHEJ repair is performed after Cas9 cuts the target position, and the process is repeated until an error occurs, which prevents Cas9-mediated DNA cleavage (Zaboikin et al, 2017)

Summary

INTRODUCTION

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-Associated (Cas) Systems. Subsequent studies showed that CRISPR and Cas endonuclease forms a complex, the gene encoding the Cas protein is located near the CRISPR locus, and that Cas creates a gap in the target DNA or RNA sequences Their genomes are protected from attack from phage nucleic acids and integrating plasmids by the CRISPR-Cas systems. The CRISPR-associated enzyme Cas breaks down the target DNA to create a DSB, the two repeated sequences are further used as templates to produce short crRNAs. Methods for DSB repair include the NHEJ and HDR pathway. The HDR pathway uses homologous donor DNA sequences from sister chromatids or foreign DNA to create accurate insertions, base substitutions between DSB sites or two DSBs, and other modifications This kind of precise modification is significant to genomic engineering in order to achieve the desired effect (Lin et al, 2014). HDR-mediated genome editing methods are limited to in vivo applications (Nami et al, 2018)

METHODS

Findings

CONCLUSIONS AND PERSPECTIVES