Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      CRISPR-Cas Systems/*genetics ; DNA/*metabolism ; Galactosyltransferases/*genetics ; Gene Editing/*methods ; RNA, Guide, CRISPR-Cas Systems/*metabolism; Animals ; Binding Sites ; DNA/genetics ; DNA Cleavage ; Galactosyltransferases/deficiency ; RNA, Guide, CRISPR-Cas Systems/chemistry ; Ribonucleoproteins/metabolism ; Swine
    • Abstract:
      The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.
      Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: BJH has an equity interest in and serves as an executive officer of Diabetes-Free, Inc, an organization that may commercially benefit from the results of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies, and does not alter our adherence to PLOS ONE policies on sharing data and materials.
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    • Accession Number:
      0 (RNA, Guide, CRISPR-Cas Systems)
      0 (Ribonucleoproteins)
      9007-49-2 (DNA)
      EC 2.4.1.- (Galactosyltransferases)
      EC 2.4.1.- (alpha-1,3-galactosyltransferase 1, porcine)
    • Publication Date:
      Date Created: 20191211 Date Completed: 20200326 Latest Revision: 20240104
    • Publication Date:
      20240104
    • Accession Number:
      PMC6903732
    • Accession Number:
      10.1371/journal.pone.0226107
    • Accession Number:
      31821359