TY  - JOUR
AU  - AlJanahi, Aisha A
AU  - Lazzarotto, Cicera R
AU  - Chen, Shirley
AU  - Shin, Tae-Hoon
AU  - Cordes, Stefan
AU  - Fan, Xing
AU  - Jabara, Isabel
AU  - Zhou, Yifan
AU  - Young, David J
AU  - Lee, Byung-Chul
AU  - Yu, Kyung-Rok
AU  - Li, Yuesheng
AU  - Toms, Bradley
AU  - Tunc, Ilker
AU  - Hong, So Gun
AU  - Truitt, Lauren L
AU  - Klermund, Julia
AU  - Andrieux, Geoffroy
AU  - Kim, Miriam Y
AU  - Cathomen, Toni
AU  - Gill, Saar
AU  - Tsai, Shengdar Q
AU  - Dunbar, Cynthia E
TI  - Prediction and Validation of Hematopoietic Stem and Progenitor Cell Off-Target Editing in Transplanted Rhesus Macaques.
JO  - Molecular therapy
VL  - 30
IS  - 1
SN  - 1525-0016
CY  - New York, NY
PB  - Nature Publ. Group
M1  - DKFZ-2021-01444
SP  - 209-222
PY  - 2022
N1  - 2022 Jan 5;30(1):209-222
AB  - 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 off-target sites predicted by CIRCLE-seq and ISP for a CD33 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/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 one 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:34174439
DO  - DOI:10.1016/j.ymthe.2021.06.016
UR  - https://inrepo02.dkfz.de/record/169364
ER  -