Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage

Jian-Ping Zhang,Jing Xu,Lu Zhang,Gary D Botimer,Ya-Wen Fu,Guo-Hua Li,David Baylink,Cameron Arakaki,Tao Cheng,Wanqiu Chen,Noah Chun,Weiping Yuan,Xiao-Lan Li,Xiao-Bing Zhang,Wei Wen

doi:10.1186/s13059-017-1164-8

Abstract

BackgroundPrecise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs).ResultsHere, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs.ConclusionsTaken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

Highlights

Precise genome editing via homology-directed repair (HDR) after double-stranded DNA cleavage facilitates functional genomic research and holds promise for gene therapy
The most commonly used system is derived from Streptococcus pyogenes (Sp), which consists of a CRISPR-associated protein-9 nuclease (Cas9) endonuclease and two separate small RNAs, called tracrRNA and crRNA [13], that can be combined with a tetraloop to form a single guide RNA [14]
We found that 20–30% HDRmediated knockin can be achieved in human induced pluripotent stem cells (iPSCs) using double cut donors with homology arms (HA) of 300–600 bp in length together with cell cycle regulators Nocodazole and CCND1

Summary

Introduction

Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. The ability to precisely edit genomes endows scientists with a powerful tool to interrogate the functionalities of any pieces of DNA in the genome of any species and it may lead to the development of new therapies that can potentially cure numerous genetic diseases [1,2,3]. Precise gene editing by homologous recombination is very inefficient, unless a DNA double-stranded break (DSB) is created at the targeting site, which increases homology-directed repair (HDR) mediated gene editing efficiency by ~1000-fold [4,5,6].

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Results

Conclusion