Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Caspase9 (Cas9) system provides a programmable technology that may be used to edit the eukaryotic genome and epigenome. CRISPR/Cas9 includes a guide RNA targeted to a gene of interest which hybridizes to a nucleotide sequence next to a protospacer-adjacent motif (PAM) which guides the Cas9 endonucleases to the target site for cleavage via double-strand breaks. A caveat of the CRISPR/Cas9 system is the creation of off-target double-strand breaks (DSBs) which may result in anomalous insertions, deletions, and translocations. Thus, assays for the sensitive detection and analysis of off-target editing are critical. Here, we describe currently available CRISPR technologies, CRISPR applications, and current analysis platforms to detect off-target effects including genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq), high-throughput genomic translocation sequencing (HTGTS), breaks labeling, enrichments on streptavidin and next-generation sequencing (BLESS), and in vitro nuclease-digested genome sequencing (Digenome-seq).
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Stephen H. Tsang receives financial support from Abeona Therapeutics, Inc and Emendo. He is also the founder of Rejuvitas and is on the scientific and clinical advisory board for Nanoscope Therapeutics.
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Xu, C.L., Ruan, M.Z., Ragi, S.D., Tsang, S.H. (2023). CRISPR Off-Target Analysis Platforms. In: Tsang, S.H., Quinn, P.M. (eds) Retinitis Pigmentosa. Methods in Molecular Biology, vol 2560. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2651-1_26
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DOI: https://doi.org/10.1007/978-1-0716-2651-1_26
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