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Development of an inducible CRISPR/Cas9 system for temporally‐modulated gene editing
Author(s) -
Olivas Micah,
Fox Doug,
Alvarez James
Publication year - 2019
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.2019.33.1_supplement.509.6
Subject(s) - crispr , cas9 , biology , genome editing , green fluorescent protein , gene knockout , gene , cancer research , microbiology and biotechnology , computational biology , genetics
Despite its prolific track record of research into primary tumor dynamics, the field of cancer biology has yet to develop a reliable methodology for interrogating the genetic profiles of recurrent tumors. CRISPR/Cas9 gene editing has proven to be foundational for recent advancements in oncogenetic screening, due in part to its high degree of specificity in achieving on‐target gene knockout while minimizing off‐target effects. The primary limitation to its application in screening pro‐recurrence factors in cancer, however, is the constitutive nature of canonical Cas9 activity, which may not be remotely regulated after the point of Cas9 translation. To address this problem, we cultivated and transduced populations of a resected invasive mammary adenocarcinoma cell type with a selection of four recombinant Cas9 transgenes and characterized the relative inducibility of each system. These Cas9's are fused to peptides whose tertiary structure renders the endonuclease inactive, restoring catalytic activity only in the presence of a fusion‐specific ligand. Populations of EGFP‐expressing adenocarcinoma cells were transduced with a modified estrogen receptor‐Cas9 fusion (ERT2‐Cas9) and assayed for target knockout efficiency upon addition of small guide GFP (sgGFP) and a Cas9‐enabling estrogen analog. After three days exposure to the analogue, flow cytometry revealed GFP expression to be markedly reduced in cells expressing ERT2‐Cas9 and minimal change in those not exposed. Once the timing and treatment of this model are refined, ERT2‐Cas9‐expressing adenocarcinoma cells will be orthotopically‐injected into mice for an in vivo screening assay. By equipping neoplastic cells with the inducible‐Cas9 machinery before the point of tumor outgrowth, it becomes possible to systematically screen pro‐survival factors at late time points in cancer progression without drastically disturbing the microenvironment of the tumor. Support or Funding Information This work was supported by ah ASPET summer research award in partnership with SURPH at Duke University. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .