In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

Abstract

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

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

“…The error rate for AmpliSeq HD is estimated to be 5-10 fold lower than for standard Illumina sequencing, but it is not zero. We applied the following criteria to score a site as truly edited in a cell sample: 1-Only INDELS were considered as edited, given that non-homologous end joining (NHEJ) very rarely results in SNPs, and most sequencing errors mimic SNPs 24,25 .…”

CRISPR-Cas off-target detection using Oxford Nanopore sequencing - is the mitochondrial genome more vulnerable to off-targets?

“…Note, previous work shows that Cas9 can bind 'with as many as nine consecutive mismatches in the sgRNA guiding' [9]. Also, Cas9 cleaves for a wide range of PAMs [16], including the NGA PAM in this case. In fact, for one gRNA (gP), in vitro studies showed that Cas9 was targeting several sites with as many as seven mismatches [16].…”

“…Note, previous work shows that Cas9 can bind 'with as many as nine consecutive mismatches in the sgRNA guiding' [9]. Also, Cas9 cleaves for a wide range of PAMs [16], including the NGA PAM in this case. (b): Frequency distrubtion of the start of the reads with respect to the mitochondrial genome (16570bp), reveals a significant number of reads begin around 9639.…”

“…Also, Cas9 cleaves for a wide range of PAMs [16], including the NGA PAM in this case. In fact, for one gRNA (gP), in vitro studies showed that Cas9 was targeting several sites with as many as seven mismatches [16]. Also, a lot of reads aligning to the mitochondrial genome (sometimes full length) are inverted after the edit.…”

Anticipating emerging biotechnology threats

“…Some of the solutions to these hidden challenges offered in the literature include (but are not limited to) using slightly shorter or slightly longer RNA guides, modifying the Cas9 enzyme to increase accuracy, using Cas9 inhibitors to reduce cleavage activity, and using novel Cas9 ortholog or other Cas enzymes. 64,67,65,74,75,76,77,78 But as demonstrated in experimental work, these solutions work well in some cases but not in others.…”