Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques
- Authors
- AlJanahi Aisha A.; Lazzarotto Cicera R.; Chen Shirley; Shin Tae-Hoon; Cordes Stefan; Fan Xing; Jabara Isabel; Zhou Yifan; Young David J.; Lee, Byung Chul; Yu Kyung-Rok; Li Yuesheng; Toms Bradley; Tunc Ilker; Hong So Gun; Truitt Lauren L.; Klermund Julia; Andrieux Geoffroy; Kim Miriam Y.; Cathomen Toni; Gill Saar; Tsai Shengdar Q.; Dunbar Cynthia E.
- Issue Date
- Jan-2022
- Publisher
- Nature Publishing Group
- Keywords
- Ca9; CRISPR; error-corrected sequencing; gene editing; gene therapy; Macaque; off-target; translocation
- Citation
- Molecular Therapy, v.30, no.1, pp 209 - 222
- Pages
- 14
- Journal Title
- Molecular Therapy
- Volume
- 30
- Number
- 1
- Start Page
- 209
- End Page
- 222
- URI
- https://scholarworks.sookmyung.ac.kr/handle/2020.sw.sookmyung/151536
- DOI
- 10.1016/j.ymthe.2021.06.016
- ISSN
- 1525-0016
1525-0024
- Abstract
- The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate offtarget sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.
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