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DISCOVER-Seq Tracks Off-target CRISPR/Cas-Gene Editing

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By Stuart P. Atkinson, Ph.D.

July 27, 2023

Introduction: The Need to Track CRISPR/Cas-mediated Gene Editing

CRISPR/Cas-mediated gene editing represents a straightforward and accurate process; however, the widespread therapeutic application of this technology still requires the precise characterization of both on- and off-target activity. Currently employed characterization methods used to detect off-target editing - such as Digenome-seq (Kim et al.), CIRCLE-seq (Tsai et al.), SITE-Seq (Cameron et al.), GUIDE-seq (Tsai et al.), or BLISS (Yan et al.) - all suffer from limitations that include the appearance of false-positives and, importantly, the inability to assess the efficiency and safety of gene editing in vivo.

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DISCOVER-Seq: A Universal and Unbiased Approach to Identifying Off-target CRISPR/Cas Activity

These problems prompted researchers from the laboratories of Beeke Wienert, Stacia K. Wyman, and Jacob E. Corn (Innovative Genomics Institute of the University of California Berkeley, USA) to develop DISCOVER-Seq (“discovery of in situ Cas off-targets and verification by sequencing”) for the unbiased identification and molecular characterization of on- and off-target CRISPR/Cas-gene editing in vitro and in vivo (Wienert et al.). The basis of DISCOVER-Seq involves monitoring the repair of Cas-induced DNA-double-stranded breaks by capturing sequences bound by endogenous DNA repair machinery factors in an endogenous chromatin context.

An initial evaluation of DNA repair proteins that might identify Streptococcus pyogenes Cas9 target sites by chromatin immunoprecipitation followed by sequencing (ChIP-seq) prompted the authors to focus on the MRE11 double-strand break repair protein. MRE11 is a subunit of the MRN complex, which functions as a cellular sensor for DNA double-strand breaks and initiates DNA damage responses. Encouragingly, ChIP-Seq with an MRE11 antibody captured the cellular activity of diverse genome-editing enzymes (Cas9 and Cas12a) at the molecular level. The subsequent combination of MRE11 ChIP-Seq with custom software (BLENDER - blunt end finder) within the DISCOVER-Seq pipeline aimed to support the in vivo identification of Cas off-target editing.

Validation steps demonstrated that DISCOVER-Seq could identify off-target editing when using well-characterized, “promiscuous” guide RNAs (“VEGFA_site2” in human K562 cells (Tsai et al.) and “Pcsk9-gP” in murine B16-F10 cells (Akcakaya et al.)) and correctly characterize guide RNAs with no off-target editing events (RNF2 and “Pcsk9-gM”) and an HBB-targeting guide with two off-target editing events (DeWitt et al.). DISCOVER-Seq also supported the identification of off-target editing and the determination of allelic specificity in Charcot-Marie-Tooth patient-derived induced pluripotent stem cells, which are often not amenable to the discovery of off-target activity due to reagent toxicity. Finally, the team explored the potential of DISCOVER-Seq in vivo in a mouse model, where it performed better compared to “verification of in vivo off-targets (VIVO),” a technique that identifies putative off-targets in purified DNA and exhaustively validates potential hits through amplicon-next generation sequencing (Akcakaya et al.).

Overall, DISCOVER-Seq represents a universal approach for the unbiased detection of off-target editing during in vivo genome editing. Furthermore, the reported performance of DISCOVER-Seq in mice and patient-derived induced pluripotent stem cells suggests the immense potential of this technique in multiple therapeutic applications, including in situ off-target discovery within individual human patient genotypes during therapeutic genome editing.

See Science, April 2019 for more on this fascinating technique, check out a recent blog post from Synthego for more information about DISCOVER-Seq, and head over to Nature Protocols for a detailed description of the experimental protocol and analysis pipeline to perform DISCOVER-seq from the same team as this original study (Wienert et al.)!

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DISCOVER-Seq+: The Next Iteration

While DISCOVER-Seq represented a considerable step forward in the field, researchers headed by Taekjip Ha (Johns Hopkins University/Howard Hughes Medical Institute, USA) sought to improve this technique even further. As MRE11 only transiently resides on DNA during active repair, the team hypothesized that the pharmacological modulation of DNA repair to improve MRE11 residence times could increase MRE11 accumulation and enhance detection sensitivity via MRE11 ChIP-seq. In their recently published paper, the team reported “DISCOVER-Seq+” as an even more sensitive method for identifying off-target genome editing in vitro and in vivo (Zou et al.).

The authors began by inhibiting one of five DNA repair inhibitors during MRE11 ChIP-qPCR assays using well-validated guide RNAs with known on- and off-target activity. Interestingly, inhibition of poly (ADP-ribose) polymerase and ATM serine/threonine kinase failed to induce any effect while DNA Ligase IV inhibition suppressed MRE11 recruitment; however, blocking non-homologous end joining (a DNA double-strand break repair mechanism) by inhibiting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (Cano et al.) significantly increased MRE11 recruitment at the target site. Overall, DNA-PKcs inhibition blocked the non-homologous end-joining repair pathway in favor of DNA double-strand break repair via MRE11-associated homology-directed repair and microhomology-mediated end-joining pathways, which boosted MRE11 residence.

DISCOVER-Seq+ displayed improved utility in three evaluated applications: i) patient-derived induced pluripotent stem cell editing, ii) generating engineered T cells for cancer immunotherapy, and iii) in vivo characterizations of CRISPR-based therapies in mouse models. Finally, in a step that takes the technique further toward human patients, the authors demonstrated that DISCOVER-Seq+ could directly measure genome-wide off-target editing in vivo in experiments targeting the PCSK9 cardiovascular risk gene in the mouse liver (Musunuru et al.).

Overall, DNA-PKcs inhibition prompted the accumulation of MRE11 at Cas target sites, which enabled DISCOVER-Seq+ to map off-target sites at a higher sensitivity than other approaches. The authors believe that DISCOVER-Seq+ will find widespread use as a state-of-the-art technology for detecting off-target editing/activity in diverse CRISPR/Cas-mediated editing applications in human patients.

For more on the development and potential of DISCOVER-Seq+, see Nature Methods, April 2023.

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About the author

Stuart P. Atkinson

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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