US 12,264,367 B2
Methods of in vivo evaluation of gene function
Xin Jin, Cambridge, MA (US); Paola Arlotta, Cambridge, MA (US); Aviv Regev, Cambridge, MA (US); Feng Zhang, Cambridge, MA (US); and Sean Simmons, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Jan. 29, 2021, as Appl. No. 17/163,015.
Application 17/163,015 is a continuation of application No. 17/025,784, filed on Sep. 18, 2020, abandoned.
Claims priority of provisional application 62/902,932, filed on Sep. 19, 2019.
Prior Publication US 2021/0172017 A1, Jun. 10, 2021
Int. Cl. C07H 21/02 (2006.01); C12N 15/90 (2006.01); C12Q 1/6883 (2018.01); C12Q 1/6897 (2018.01)
CPC C12Q 1/6883 (2013.01) [C12N 15/907 (2013.01); C12Q 1/6897 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01); C12Q 2600/136 (2013.01)] 24 Claims
 
1. A method of identifying functions of a plurality of target genes in parallel in vivo, comprising:
a. introducing, in vivo, a plurality of genetic perturbations in each of a plurality of progenitor cells in a Cas animal model, wherein the plurality of genetic perturbations are introduced by delivering a pool of guide RNAs (gRNAs) to the plurality of progenitor cells,
wherein the pool of gRNAs comprises two or more different gRNAs that bind each target gene of the plurality of target genes,
wherein the two or more different gRNAs for each target gene are operatively coupled to a reporter gene and a unique barcode corresponding the perturbation identity,
b. generating an enriched perturbed cell population by enriching for cells expressing the reporter gene;
c. identifying cell types and corresponding perturbations via scRNA-seq in the enriched perturbed cell population; and
d. detecting one or more target gene modules that co-vary within a cell type in the enriched perturbed cell population.