The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment

(Pages: 532-539)
Mohsen Basiri, Ph.D, 1,2Mehrdad Behmanesh, Ph.D, 1,*Yaser Tahamtani, Ph.D, 2Keynoosh Khalooghi, M.Sc, 2Azadeh Moradmand, M.Sc, 2Hossein Baharvand, Ph.D, 2,3,*
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Department of Developmental Biology, University of Science and Culture, Tehran, Iran
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Department of Developmental Biology, University of Science and Culture, Tehran, Iran
*Corresponding Addresses: P.O.Box: 14115-154 Department of Genetics Faculty of Biological Sciences Tarbiat Modares University Tehran Iran P.O. Box: 16635-148 Department of Stem Cells and Developmental Biology Cell Science Research Center Royan Institute for Stem Cell Biology and Technology ACECR Tehran Iran Emails:behmanesh@modares.ac.ir,baharvand@royaninstitute.org
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Basiri Mohsen, Behmanesh Mehrdad, Tahamtani Yaser, Khalooghi Keynoosh, Moradmand Azadeh, Baharvand Hossein. The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment. Cell J. 2017; 18(4): 532-539.

Abstract

Objective

CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid.

Materials and Methods

In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs).

Results

Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus.

Conclusion

While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.