Journal Article

DKFZ-2021-01444

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Prediction and Validation of Hematopoietic Stem and Progenitor Cell Off-Target Editing in Transplanted Rhesus Macaques.

AlJanahi, A. A. ; Lazzarotto, C. R. ; Chen, S. ; Shin, T.-H. ; Cordes, S. ; Fan, X. ; Jabara, I. ; Zhou, Y. ; Young, D. J. ; Lee, B.-C. ; Yu, K.-R. ; Li, Y. ; Toms, B. ; Tunc, I. ; Hong, S. G. ; Truitt, L. L. ; Klermund, J. ; Andrieux, G.DKFZ* ; Kim, M. Y. ; Cathomen, T. ; Gill, S. ; Tsai, S. Q. ; Dunbar, C. E.

2022
Nature Publ. Group New York, NY

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Please use a persistent id in citations: doi:10.1016/j.ymthe.2021.06.016

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 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.

Note: 2022 Jan 5;30(1):209-222

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Contributing Institute(s):

1. DKTK FR zentral (FR01)

Research Program(s):

1. 899 - ohne Topic (POF4-899) (POF4-899)

Appears in the scientific report 2021

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Database coverage:
; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; Ebsco Academic Search ; Essential Science Indicators ; IF >= 10 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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