Journal
FEBS LETTERS
Volume 591, Issue 6, Pages 903-913Publisher
WILEY
DOI: 10.1002/1873-3468.12599
Keywords
biallelic genome engineering; CRISPR/Cas9; donor plasmid; homology directed repair; surrogate reporter
Funding
- National Science and Technology Major Project of China [2014ZX0801009B]
- Postdoctoral Science Foundation of Shaanxi Province of China [2016BSHEDZZ122]
- Scientific and Technological Project of Shaanxi Province of China [2014K02-07-01]
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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome-modified cells is often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporter cassettes into paired donor plasmids, which were used as both the surrogate reporters and the knock-in donors. By applying our dual surrogate reporter-integrated donor system, we demonstrate high frequency of CRISPR/Cas9-mediated biallelic genome integration in both human HEK293T and porcine PK15 cells (34.09% and 18.18%, respectively). Our work provides a powerful genetic tool for assisting the selection and enrichment of cells with targeted biallelic genome modification.
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