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Multidimensional chemical control of CRISPR‐Cas9
Author(s) -
Moore Christopher L,
Maji Basudeb,
Choudhary Amit,
Shoulders Matt
Publication year - 2017
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.31.1_supplement.909.5
Subject(s) - crispr , genome editing , cas9 , computational biology , biology , gene , regulation of gene expression , endonuclease , genetics
Facile Cas9‐derived technologies have revolutionized the gene targeting landscape for functions ranging from DNA editing to transcriptional regulation. However, a critical need still exists for robust methods to regulate the activities of these endonuclease‐based tools in living systems. In the context of gene editing, small molecule‐based tools could be utilized to regulate the levels and/or the activity of endonucleases to enhance specificity and provide temporal control of gene editing. For gene regulation, complete temporal control of transcription induction is essential for the study cell signaling, stress response pathways and other complex biological systems. Furthermore, dosable control of transcriptional activity is absolutely essential to ensure that physiologically relevant levels of gene expression are achieved. In order to address these technical needs, we designed a broadly applicable method for controlling applied activities of RNA‐guided nuclease biology by integrating destabilized domain technology with the CRISPR/Cas9 system. We find that incorporation of destabilized domain and CRISPR/Cas9 transcriptional perturbation technologies provides users with a convenient method for controlling many aspects, including time and extent of endogenous gene upregulation, through addition or removal of a small molecule. For gene editing, we find that destabilized domain Cas9 confers provides users with exquisite control parameters and reduces off‐target effects. Support or Funding Information This work was supported by the NIH (Director's New Innovator Award 1DP2GM119162 to M.D.S.; grant 1R21AI126239‐01 to A.C.), the Edward Mallinckrodt, Jr. Foundation (Faculty Scholar Award to M.D.S.), and the Burroughs Wellcome Fund (Career Award at the Scientific Interface to A.C.). C.L.M. acknowledges the National Science Foundation for a Graduate Research Fellowship.We achieved small‐molecule control of transcriptional activation and genome editing by tethering inducible protein degron domains (destabilized domains) to an engineered CRISPR–Cas9 system.( a ) Top, small‐molecule‐mediated transcription induction via a destabilized domain‐fused transcription activation domain (DHFR.PP7.VP64), dSpCas9, and an sgRNA. Bottom, HEK293T cells transfected with to regulated IL1RN transcription. ( b ) Rapid turn‐off of transcription targeted to IL1RN or NANOG . ( c ) Independent, small‐molecule‐mediated control of transcript expression for two genes ( d ) Highly dose‐responsive endogenous gene upregulation in cells when targeting to IL1RN and/or ASCL1.( a , b ) TMP‐ and 4OHT‐dose‐dependent control of on‐ and off‐target activity of destabilized domain‐regulated Cas9 targeting VEGFA ( a ) or EMX1 ( b ). ( c , d ) Ratiometric representation of on‐target:off‐target indel frequencies of DD.SpCas9.DD for VEGFA ( c ) and EMX1 ( d ). ( e ) Temporal control of DHFR.SpCas9.DHFR‐mediated genome editing analyzed by an eGFP disruption assay. Error bars represent ±s.e.m. from biological replicates ( n = 4 ( a – d ) or 5 ( e )).