Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

Abstract

As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.

Comparative analysis of CRISPR off-target activity discovery tools followingex vivoediting of CD34+hematopoietic stem and progenitor cells

“…5b ). Additionally, unlike prior studies that relied on a pre-existing pool of cells with oncogenic mutations 36,37 , by conducting our experiments in primary HSPCs that do not harbor such aberrations 38 , it is not surprising that this study found no bona fide OT sites residing in exons of known tumor suppressors or oncogenes. This was further confirmed by a recent publication that found no evidence that Cas9 introduced or enriched for oncogenic mutations following ex vivo editing in primary HSPCs 38 .…”

“…Additionally, unlike prior studies that relied on a pre-existing pool of cells with oncogenic mutations 36,37 , by conducting our experiments in primary HSPCs that do not harbor such aberrations 38 , it is not surprising that this study found no bona fide OT sites residing in exons of known tumor suppressors or oncogenes. This was further confirmed by a recent publication that found no evidence that Cas9 introduced or enriched for oncogenic mutations following ex vivo editing in primary HSPCs 38 . Taken together, our study highlights the importance of evaluating Cas9 OT detection tool performance in the appropriate application-specific context (e.g., high-fidelity nuclease, primary HSPCs) where epigenetic factors and DNA damage repair mechanisms can impact the appearance of unwanted OT editing.…”

“…Though datasets for empirical methods were not available for many of the guides used in this study, we found no OT sites validated under high-fidelity editing conditions identified solely by empirical methods. While in silico methods appear capable of capturing most true OT events, we note that they rely on homology to a consensus genome and therefore would not take into account patient-specific variation, which has been shown to impact Cas9 activity 38 . The empirical methods, however, are also limited by not being performed on a diverse array of genomes and are usually also performed on a single genome.…”

CRISPR nuclease off-target activity and mitigation strategies

“…These methods vary widely in their approach and even starting material, using cell-free genomic DNA in vitro ( Kim et al, 2015 ; Cameron et al, 2017 ; Tsai et al, 2017 ; Kim and Kim, 2018 ; Lazzarotto et al, 2020 ; Dobbs et al, 2022 ), in intact live cells ex vivo ( Crosetto et al, 2013 ; Tsai et al, 2015 ; Yan et al, 2017 ; Wienert et al, 2019 , 2020 ; Zhu et al, 2019 ), and in vivo animal models ( Akcakaya et al, 2018 ; Wienert et al, 2019 ; Liang et al, 2022 ). Methods that use cell- and nucleosome-free DNA generally report the highest number of off-targets, many of which cannot be verified in a cellular context ( Cromer et al, 2022a ). Furthermore, methods such as GUIDE-Seq have been shown to identify more off-target sites in immortalized cell lines than when assaying primary cells ( Shapiro et al, 2020 ).…”

Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

“…(3) Evaluating indels alone may also miss complex DNA damage outcomes such as large deletions and translocations [8,39]. Furthermore, deep amplicon sequencing may miss rarer off-target sites due to a lack of enrichment of altered DNA molecules and insufficient sequencing depth [40]. For all these reasons, enrichment for sites with evidence of CRISPR-induced DNA damage is a more holistic readout of offtarget activity than indels alone.…”