In vivo CRISPR editing with no detectable genome-wide off-target mutations
| Autor: | Frank Seeliger, Jimmy A. Guo, Luca Pinello, Mohammad Bohlooly-Y, Roberto Nitsch, Lorenz M. Mayr, Shengdar Q. Tsai, Mick D. Fellows, Pinar Akcakaya, Marcello Maresca, Jose Malagon-Lopez, Alba Carreras, Mikael Bjursell, J. Keith Joung, Nhu T. Nguyen, Mike Firth, Sara P. Garcia, Kendell Clement, Martin J. Aryee, Michelle J. Porritt, Tania Baccega, Maggie L. Bobbin |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Male
0301 basic medicine CRISPR-Associated Proteins Computational biology Biology Genome Article Substrate Specificity Mice 03 medical and health sciences PCSK9 Gene INDEL Mutation Genome editing In vivo Animals Humans CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Transgenes Guide RNA Gene Editing Multidisciplinary Highly sensitive Mice Inbred C57BL 030104 developmental biology Mutation Female Proprotein Convertase 9 CRISPR-Cas Systems |
| Zdroj: | Nature |
| ISSN: | 1476-4687 0028-0836 |
| Popis: | CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1–6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing. A strategy developed to define off-target effects of gene-editing nucleases in whole organisms is validated and leveraged to show that CRISPR–Cas9 nucleases can be used effectively in vivo without inducing detectable off-target mutations. |
| Databáze: | OpenAIRE |
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