US 12,139,705 B2
Deep learning enabled spatial optical barcodes for pooled library screens
Morgan S. Schwartz, Pasadena, CA (US); David A. Van Valen, Pasadena, CA (US); and Edward Pao, San Dimas, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by California Institute of Technology, Pasadena, CA (US)
Filed on Nov. 4, 2020, as Appl. No. 17/088,594.
Claims priority of provisional application 62/930,343, filed on Nov. 4, 2019.
Prior Publication US 2021/0130810 A1, May 6, 2021
Int. Cl. C12N 15/10 (2006.01); C12N 15/11 (2006.01); G06T 7/00 (2017.01); G06V 10/764 (2022.01); G06V 20/69 (2022.01); G16B 40/00 (2019.01)
CPC C12N 15/1065 (2013.01) [C12N 15/11 (2013.01); G06T 7/0012 (2013.01); G06V 10/764 (2022.01); G06V 20/695 (2022.01); G06V 20/698 (2022.01); G16B 40/00 (2019.02); C12N 2310/20 (2017.05); G06T 2207/20081 (2013.01); G06T 2207/30024 (2013.01); G06T 2207/30072 (2013.01)] 21 Claims
 
1. A method for determining perturbations in a plurality of single cells, the method comprising:
expressing in each of a plurality of single cells (i) a plurality of guide ribonucleic acids (gRNAs) comprising a perturbation gRNA (AgRNA) and one or more barcode gRNAs, and (ii) a dead CRISPR associated protein 9 (dCas9), wherein the barcode gRNAs each comprises a barcode region comprising one or more optical detection probe binding sites for a plurality of optical detection probes, thereby the AgRNA binds to a first dCas9 and the first dCas9 bound to the AgRNA binds to a chromosome sequence of a genome of a single cell, resulting in a perturbation in the single cell, and thereby each of the barcode gRNAs binds to a second dCas9 and the second dCas9 bound to the barcode gRNA binds a predetermined spatial region of the genome of the single cell, wherein presence of a combination, of (i) each of the optical detection probe binding sites in the barcode regions of the barcode gRNAs expressed in the single cell and (ii) the corresponding predetermined spatial region where the second dCas9 bound to the barcode gRNA comprising the barcode region binds to, indicates the AgRNA is expressed in the single cell;
staining the plurality of single cells with the plurality of optical detection probes, wherein two optical detection probes of the plurality of optical detection probes comprise different optical labels and are capable of binding to two optical detection probe binding sites, of the barcode gRNAs expressed in the plurality of single cells, comprising different sequences, thereby optical detection probes of the plurality of optical barcodes bind to the optical detection probe binding sites of the barcode gRNAs expressed in the plurality of single cells;
imaging the plurality of single cells comprising the optical detection probes bound to the optical detection probe binding sites of the barcode gRNAs expressed in the plurality of single cells to generate a plurality of images; and
analyzing the plurality of images generated to determine, for each of the plurality of single cells, the combination of optical detection probe binding sites expressed in the single cell and corresponding predetermined spatial regions, thereby determining the perturbation in the single cell.