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

Abstract

Background After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. Results We 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%. Conclusions Timely 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.

CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

“…In the HepG2 Δi50 deletion clone, we detected a large genomic deletion. Such deletions are the consequence of the Cas9 systems and can occur at different genomic loci and in many cell lines with various frequency (69,(72)(73)(74). Since the size of the large deletion is atypical for the NHEJ repair machinery (75,76), other repair mechanisms such as MMEJ could play a role (69,77,78).…”

Improved and Flexible HDR Editing by Targeting Introns in iPSCs

“…Albacore (version 2.3.1, Oxford Nanopore Technologies) transformed raw fast5 data into bases and quality scores. Then, we processed the data as described previously [ 28 , 29 ]. In brief, sequencing adapters were removed by Porechop [ 30 ] (version 0.2.4) and then processed with Seqkit to grep for individual reads of PCR products.…”

CRISPR–Cas9 gene editing induced complex on-target outcomes in human cells

“…However, because of the intrinsic limitations of long-range PCR, megadeletions >10 kb have not been fully illustrated, and their occurrence may depend on the unique features of the targets. That said, deletions exceeding 1 kb are observed at considerably lower frequencies than shorter deletions (100−500 bp) [49].…”

“…Of interest, HDR outcompetes MMEJ [47], likely because the presence of long homology in HDR stabilizes the annealing of donor template and the DSB-proximal genome sequence. As such, inhibition of the NHEJ pathway increases the chances of being repaired by MMEJ, leading to more deletions, small and large [48,49]. Similarly, inhibition of the NHEJ pathway is a common strategy to increase HDR efficiency [25,50,51].…”