CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.

ON-target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.

Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.

Genome editing in large animal models.

β-thalassemia and sickle cell disease (SCD) are the most common monogenic diseases in the world and are potentially curable after allogeneic hematopoietic stem cell transplantation (HSCT) or autologous HSCT after genetic modification. Autologous gene therapy has the potential to offer a universal cure that overcomes many limitations of allogeneic HSCT including the lack of available donors, graft-vs-host disease, and graft rejection. Significant progress in gene therapy for the hemoglobinopathies has been made over the last several decades, now with multiple ongoing clinical trials investigating both gene addition and gene-editing strategies. Available results from a small number of patients, some with relatively short follow-up, are promising, with current efforts focused on continuing to improve the efficacy, durability, and safety of gene therapies for the cure of hemoglobin disorders. 

Gene Therapy for Hemoglobinopathies: Beta-Thalassemia, Sickle Cell Disease.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures. 

Error-corrected Next Generation Sequencing - Promises and Challenges for Genotoxicity and Cancer Risk Assessment.

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

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 in vitro off-target prediction method, more accurately identifies off-targets compared to 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 guide RNAs designed against TET2 and CD33 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 CD33 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 in vivo 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

https://www.biorxiv.org/content/biorxiv/early/2020/07/05/2020.07.05.186858.full.pdf

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

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

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