RT Journal Article
SR Electronic
T1 Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
JF Science
JO Science
FD American Association for the Advancement of Science
SP 286
OP 289
DO 10.1126/science.aav9023
VO 364
IS 6437
A1 Wienert, Beeke
A1 Wyman, Stacia K.
A1 Richardson, Christopher D.
A1 Yeh, Charles D.
A1 Akcakaya, Pinar
A1 Porritt, Michelle J.
A1 Morlock, Michaela
A1 Vu, Jonathan T.
A1 Kazane, Katelynn R.
A1 Watry, Hannah L.
A1 Judge, Luke M.
A1 Conklin, Bruce R.
A1 Maresca, Marcello
A1 Corn, Jacob E.
YR 2019
UL http://science.sciencemag.org/content/364/6437/286.abstract
AB Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice.Science, this issue p. 286, p. 289, p. 292; see also p. 234CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.