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

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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 Pdx1 ^GFP 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.

Related collections

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Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Kai Li, Gang Wang, Troels Andersen … (2014)

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.

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Author and article information

Journal

Journal ID (nlm-ta): Cell J

Journal ID (iso-abbrev): Cell J

Journal ID (publisher-id): Royan Institute

Title: Cell Journal (Yakhteh)

Publisher: Royan Institute

ISSN (Print): 2228-5806

ISSN (Electronic): 2228-5814

Publication date (Print): Winter 2017

Publication date (Electronic): 26 September 2016

Volume: 18

Issue: 4

Pages: 532-539

Affiliations

[1 ]Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

[2 ]Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

[3 ]Department of Developmental Biology, University of Science and Culture, Tehran, Iran

Author notes

[*Corresponding Addresses: ]P.O.Box: 14115-154Department of GeneticsFaculty of Biological SciencesTarbiat Modares University TehranIranP.O. Box: 16635-148Department of Stem Cells and Developmental BiologyCell Science Research CenterRoyan Institute for Stem Cell Biology and Technology ACECRTehranIran Emails: behmanesh@ 123456modares.ac.ir , baharvand@ 123456royaninstitute.org

Article

Other ID: Cell-J-18-532

DOI: 10.22074/cellj.2016.4719

PMC ID: 5086331

PubMed ID: 28042537

SO-VID: 0d52d32f-705b-4620-b459-5e8f0a13ac07

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 7 October 2015

Date accepted : 29 February 2016

Custom metadata

Citation Basiri M, Behmanesh M, Tahamtani Y, Khalooghi K, Moradmand A, Baharvand H. 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.

Keywords: crispr-cas systems,gene targeting,embryonic stem cells,pdx1

Data availability:

Keywords: crispr-cas systems, gene targeting, embryonic stem cells, pdx1