Precise and efficient scarless genome editing in stem cells using CORRECT
| Autor: | Shaun Teo, Dylan Kwart, Marc Tessier-Lavigne, Dominik Paquet |
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| Rok vydání: | 2017 |
| Předmět: |
Pluripotent Stem Cells
0301 basic medicine Mutagenesis (molecular biology technique) Biology Transfection medicine.disease_cause Genome General Biochemistry Genetics and Molecular Biology Cell Line 03 medical and health sciences 0302 clinical medicine Genome editing medicine Humans CRISPR Gene Knock-In Techniques Induced pluripotent stem cell Gene Editing Genetics Mutation Base Sequence Genome Human Cas9 Flow Cytometry 030104 developmental biology CRISPR-Cas Systems Stem cell 030217 neurology & neurosurgery |
| Zdroj: | Nature Protocols. 12:329-354 |
| ISSN: | 1750-2799 1754-2189 |
| DOI: | 10.1038/nprot.2016.171 |
| Popis: | CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells. This protocol outlines in detail how to implement either the re-Guide or re-Cas variants of CORRECT to generate scarlessly edited isogenic stem cell lines with intended monoallelic and biallelic sequence changes in ∼3 months. |
| Databáze: | OpenAIRE |
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