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US 2018/0023139 A1Published application
Methods of identifying essential protein domains
AssigneeCold Spring Harbor Laboratory
InventorsChristopher H. Vakoc, Junwei Shi, Justin B. Kinney
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Abstract
Provided herein, in some aspects, are methods of determining whether a candidate protein, more specifically a functional domain of a candidate protein, is essential for viability of cells of interest using clustered regularly interspaced short palindromic repeat (CPJSPR)-Cas9 technology which holds great promise for genetic screening and for the discovery of therapeutic targets.
Claims20
A method of determining whether a functional domain of a candidate protein is essential for viability of cells of interest, comprising:
- (a) introducing, into a population of Cas9-expressing cells of interest, a nucleic acid encoding a single guide RNA (sgRNA) that targets a first region of an gene encoding a candidate protein, wherein the first region encodes a functional domain of the candidate protein, thereby producing a first population of cells comprising a subpopulation of cells that comprise Cas9 nuclease and sgRNA that targets the first region;
- (b) introducing, into a population of Cas9-expressing cells of interest, a nucleic acid encoding a sgRNA that targets a second region of a gene encoding the candidate protein, wherein the second region is 5′ to the first region and does not encode a functional domain of the candidate protein, thereby producing a second population of cells comprising subpopulation of cells that comprise Cas9 nuclease and sgRNA that targets the second region;
- (c) culturing the first population of cells produced in (a) and the second populations of cells produced in (b) under conditions that result in CRISPR-induced indel mutagenesis of the first region and of the second region, thereby producing a first population of cultured cells and a second population of cultured cells;
- (d) assessing the normalized percentage of sgRNA-positive cells (NP) over time in the first population of cultured cells to determine a decrease over time in the NP for the first population of cultured cells;
- (e) assessing the NP over time in the second population of cultured cells to determine a decrease over time in the NP for the second population of cultured cells;
- and
- (f) comparing the decrease in NP for the first population (ΔNP1) to the decrease in NP for the second population (ΔNP2), wherein if ΔNP1 is greater than ΔNP2, the functional domain of the candidate protein is essential for viability of cells of interest.
4 more elements
A method of determining whether a functional domain of a candidate protein is essential for viability of cells of interest, comprising:
- (a) introducing, into a subpopulation of a population of Cas9-expressing cells of interest, a nucleic acid encoding a single guide RNA (sgRNA) that targets a first region of a gene encoding a candidate protein, wherein the first region encodes a functional domain of the candidate protein, thereby producing a first population of cells comprising a subpopulation of cells that comprise Cas9nuclease and sgRNA that targets the first region;
- (b) introducing, into a subpopulation of a population of Cas9-expressing cells of interest, a nucleic acid encoding a sgRNA that targets a second region of a gene encoding the candidate protein, wherein the second region is 5′ to the first region and does not encode a functional domain of the candidate protein, thereby producing a second population of cells comprising subpopulation of cells that comprise Cas9 nuclease and sgRNA that targets the second region;
- (c) culturing the first population of cells produced in (a) and the second populations of cells produced in (b) under conditions that result in CRISPR-induced indel mutagenesis of the first region and of the second region, thereby producing a first population of cultured cells and a second population of cultured cells;
- (d) assessing the normalized percentage of CRISPR-induced indel mutations (NP) over time in the first population of cultured cells to determine a decrease over time in the NP for the first population of cultured cells;
- (e) assessing the NP over time in the second population of cultured cells to determine a decrease over time in the NP for the second population of cultured cells;
- and
- (f) comparing the decrease in NP for the first population (ΔNP1) to the decrease in NP for the second population (ΔNP2), wherein if ANP1 is greater than ΔNP2, the functional domain of the candidate protein is essential for viability of cells of interest.
4 more elements
Dependencies are inferred from the claim text.