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A Small Molecule Enhanced CRISPR/Cas9 Methodology for Genome Editing of Dog ( Canis familiaris) induced Pluripotent Stem Cells (diPSC)
Author(s) -
Federico Francesca,
Federico Niksha,
Sternik Gabriel,
Federico Tessya,
EsmaeliAzad Babak
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.571.6
Subject(s) - crispr , genome editing , computational biology , genome , cas9 , biology , induced pluripotent stem cell , forward genetics , gene , homology directed repair , genetics , embryonic stem cell , dna repair , dna mismatch repair
Bacterial CRISPR‐Cas9 system has emerged as an effective tool for sequence‐specific gene knock‐out through non‐homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences (gene knock‐in) via homology‐directed repair (HDR). Because of this a long and tedious screening process via cell sorting or selection, expansion, and sequencing is often required to identify correctly edited cells. Improving the efficiency of precise CRISPR gene editing remains a major challenge for many applications, such as generating sufficient numbers of genome‐edited animals or genome edited patient reprogrammed cell lines for disease modeling. Using our patented technology to develop Artificial 3D Microenvironment Niche (US Patent # 904737), we have developed a reporter based screening approach for high‐throughput identification of chemical compounds that can modulate precise genome editing through HDR. In preliminary experiments, we have identified small molecule classes that enhance CRISPR mediated HDR efficiency more than 9 fold. Additionally, we have found that small molecules that inhibit HDR, enhance insertion and deletion mutations via NHEJ and “vice versa”. We plan to further test this refined genome editing methodology for growth hormone gene modifications in dog ( Canis familiaris) induced pluripotent stem cells (diPSC), attempting to correct genetically inherited disorders in dogs and for the development of “designer pets” of the future. Our preliminary findings with this experimental system will be presented. Support or Funding Information This project was supported by Internal R&D funds from DNAmicroarray, Inc.