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

Aisha A. AlJanahi, Cicera R. Lazzarotto, Shirley Chen, Tae Hoon Shin, Stefan Cordes, Xing Fan, Isabel Jabara, Yifan Zhou, David J. Young, Byung Chul Lee, Kyung Rok Yu, Yuesheng Li, Bradley Toms, Ilker Tunc, So Gun Hong, Lauren L. Truitt, Julia Klermund, Geoffroy Andrieux, Miriam Y. Kim, Toni CathomenSaar Gill, Shengdar Q. Tsai, Cynthia E. Dunbar

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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 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.

Original languageEnglish
Pages (from-to)209-222
Number of pages14
JournalMolecular Therapy
Volume30
Issue number1
DOIs
StatePublished - Jan 5 2022

Keywords

  • CRISPR
  • Ca9
  • Macaque
  • error-corrected sequencing
  • gene editing
  • gene therapy
  • off-target
  • translocation

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