Validation and Long-Term Follow Up of CD33 Off-Targets Predicted In Vitro and In Silico Using Error-Corrected Sequencing in Rhesus Macaques

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 ask whether CIRCLE-Seq (CS), an <jats:italic>in vitro</jats:italic> off-target prediction method, more accurately identifies off-targets compared to <jats:italic>in silico</jats:italic> 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 guide RNAs designed against <jats:italic>TET2</jats:italic> and <jats:italic>CD33</jats:italic> genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a <jats:italic>CD33</jats:italic> gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior <jats:italic>in vivo</jats:italic> in animals followed for up to 2 years. In conclusion, neither methodology 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.</jats:p>

CD7-deleted hematopoietic stem cells can restore immunity after CAR T cell therapy

“…We did not find any off-target editing primary human CD34 + cells in any of the predicted off-target sites that we queried, despite high levels of on-target mutations. These results are consistent with other studies investigating CRISPR/Cas9 off-target activity in HSCs (25)(26)(27), where minimal off-target events were detected as long as the gRNA was chosen carefully. However, this does not preclude the possibility of off-target sites unique to HSCs that were not identified through our prediction models, or of off-target editing specific to individuals with certain single-nucleotide variations.…”