-
Tytuł:
-
Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques
-
Autorzy:
-
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.
-
Źródło:
-
Molecular Therapy; 20210101, Issue: Preprints
-
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 vitrooff-target prediction method, with in silicoprediction (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 CD33guide 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 vivoin 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.