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Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.
Fu, Ya-Wen; Dai, Xin-Yue; Wang, Wen-Tian; Yang, Zhi-Xue; Zhao, Juan-Juan; Zhang, Jian-Ping; Wen, Wei; Zhang, Feng; Oberg, Kerby C; Zhang, Lei; Cheng, Tao; Zhang, Xiao-Bing.
Affiliation
  • Fu YW; 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.
  • Dai XY; 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.
  • Wang WT; 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.
  • Yang ZX; 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.
  • Zhao JJ; 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.
  • Zhang JP; 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.
  • Wen W; 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.
  • Zhang F; 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.
  • Oberg KC; Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA 92350, USA.
  • Zhang L; 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.
  • Cheng T; CAMS Key Laboratory of Gene Therapy for Blood Diseases, Tianjin 300020, China.
  • Zhang XB; Tianjin Laboratory of Blood Disease Gene Therapy, Tianjin 300020, China.
Nucleic Acids Res ; 49(2): 969-985, 2021 01 25.
Article in En | MEDLINE | ID: mdl-33398341
Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Ribonucleoproteins / Parvovirinae / DNA End-Joining Repair / Recombinational DNA Repair / CRISPR-Cas Systems / Gene Editing / Genetic Vectors Limits: Adult / Humans Language: En Journal: Nucleic Acids Res Year: 2021 Document type: Article Affiliation country: China Country of publication: United kingdom

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Ribonucleoproteins / Parvovirinae / DNA End-Joining Repair / Recombinational DNA Repair / CRISPR-Cas Systems / Gene Editing / Genetic Vectors Limits: Adult / Humans Language: En Journal: Nucleic Acids Res Year: 2021 Document type: Article Affiliation country: China Country of publication: United kingdom