Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion

Wei Wen,Si-Ang Li,Zhi-Xue Yang,Jian-Ping Zhang,Xiao-Bing Zhang,Feng Zhang,Ya-Wen Fu,Meng-Di Yin,Zi-Jun Quan,Mei Zhao,Jing Xu,Tao Cheng,Guo-Hua Li

doi:10.1186/s13059-021-02462-4

Abstract

BackgroundAfter repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.ResultsWe show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%.ConclusionsTimely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

Highlights

After repairing double-strand breaks (DSBs) caused by Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences
For the first time, we show that associated virus serum type 6 (AAV6) donor-mediated homology-directed repair (HDR) almost abrogates, and non-homologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertion attenuates large-fragment deletions, providing solutions to this type of adverse effect that hampers the clinical translation of genome editing-based therapy
Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions in multiple cell types To identify significant genetic changes after CRISPR-mediated double-stranded DNA (dsDNA) cleavage (Fig. 1a), we PCR-amplified a 4- to 6-kb region flanking the Cas9-gRNA target sites at EEF2, AAVS1, and two BCL11A loci of three cell types (Additional file 1: Fig. S1a)

Summary

Introduction

After repairing double-strand breaks (DSBs) caused by CRISPR-Cas cleavage, genomic damage, such as large deletions, may have pathogenic consequences. Guided DNA endonuclease system that targets specific genomic sequences [1]. Genome editing via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) after CRISPR-mediated double-stranded DNA (dsDNA) cleavage has transformed the field of cell and gene therapy. Besides off-target effects, a combination of long-range PCR and third-generation sequencing technologies has led to the identification of frequent large fragment deletions (kilobase scale) and even complex genomic rearrangements at target sites of gene-edited cells and human embryos [9,10,11,12,13]. Affordability, and speed in data production make it suitable for a comprehensive investigation of genome-editing associated large deletions [15]

Methods

Results

Discussion

Conclusion