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

  • Genome Biol. 2021 Aug 20;22(1):236. doi: 10.1186/s13059-021-02462-4.
Wei Wen   #  1 ,  Zi-Jun Quan   #  1 ,  Si-Ang Li   #  1 ,  Zhi-Xue Yang  1 ,  Ya-Wen Fu  1 ,  Feng Zhang  1 ,  Guo-Hua Li  1 ,  Mei Zhao  1 ,  Meng-Di Yin  1 ,  Jing Xu  1 ,  Jian-Ping Zhang  1 ,  Tao Cheng  2  3  4 ,  Xiao-Bing Zhang  5
Affiliations
  • 1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China.
  • 2. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China. [email protected].
  • 3. Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. [email protected].
  • 4. Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. [email protected].
  • 5. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China. [email protected].
  • # Contributed equally.
PMID: 34416913 DOI: 10.1186/s13059-021-02462-4
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.

Keywords
CRISPR-Cas9; Genome editing; Hematopoietic stem and progenitor cells (HSPCs); Homology-directed repair (HDR); Induced pluripotent stem cells (iPSCs); Large fragment deletions; Nanopore sequencing; Non-homologous end joining (NHEJ); T cells.
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