title: The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment.
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.
  
  
  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). UsingPdx1gene 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 ofPdx1GFPknock-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Basiri
authors.affiliation: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Behmanesh
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Yaser
authors.initials: Y
authors.lastname: Tahamtani
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Keynoosh
authors.initials: K
authors.lastname: Khalooghi
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Moradmand
authors.affiliation: 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.
authors.forename: Hossein
authors.initials: H
authors.lastname: Baharvand
keywords: CRISPR-Cas Systems; Embryonic Stem Cells; Gene Targeting; Pdx1