Journal
NATURE PROTOCOLS
Volume 12, Issue 2, Pages 329-354Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.171
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Funding
- Rockefeller University
- New York Stem Cell Foundation
- Ellison Foundation
- Cure Alzheimer's Fund
- Empire State Stem Cell fund through New York State Department of Health
- National Sciences and Engineering Research Council of Canada
- German Academy of Sciences Leopoldina
- Agency for Science, Technology and Research of Singapore
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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 similar to 3 months.
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