Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques

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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
Nature Publishing Group
Molecular Therapy, Vol.30 No.1, pp.209-222
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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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Journal Papers (저널논문_농생명공학부)
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