US 2021/0340527 A1 · ENCODING OF DNA VECTOR IDENTITY VIA ITERATIVE HYBRIDIZATION DETECTION OF A BARCODE TRANSCRIPT

Abstract

Embodiments disclosed herein are directed to a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH—in conjunction with a large array of genetic perturbations applied as a pool in a single test volume.

Claims36

Dependent claims

1-36. (canceled)

A method for screening cells for comprising:

culturing a cell or population of cells in one or more discrete volumes;
a) introducing one or more vectors into the cell or cell population, each vector comprising nucleic acid sequences encoding
i) one or more optical barcodes, each optical barcode comprises a set of ordered segments, each segment comprising a nucleic acid base sequence from a set of possible nucleic acid bases or sequences for that particular segment;
and
ii) one or more genetic perturbations;
thereby introducing the one or more optical barcodes and the one or more genetic perturbations into the cell;
b) incubating the cells to allow for expression of RNA comprising the one or more optical barcodes;
and
c) detecting the one or more optical barcodes in the RNA to identify the one or more genetic perturbations present in the cell or cell populations.

6 more elements

Dependencies are inferred from the claim text.

Facts

Application

17/321,282Filed 14 May 2021

Priority

16 Mar 2015

Published

4 Nov 2021

Term ends

Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12N 15/1065
Isolating an individual clone by screening libraries: preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis,…
C12N 15/102
Processes for the isolation, preparation or purification of DNA or RNA: mutagenizing nucleic acids
C12N 15/1079
Isolating an individual clone by screening libraries: screening libraries by altering the phenotype or phenotypic trait of the host
C12N 15/1082
Isolating an individual clone by screening libraries: preparation or screening gene libraries by chromosomal integration of polynucleotide…

Family

Application62/133,821Us provisional application · filed 16 Mar 2015

Application62/133,539Us provisional application · filed 16 Mar 2015

Application15/558,504Continuation · filed 14 Sep 2017

Application17/321,282Continuation

US 11,046,952 B2Related patent

US 2018/0080019 A1Published application

US 2021/0340527 A1This patent

From the US filing history. Foreign family members aren’t listed.

Encoding of dna vector identity via iterative hybridization detection of a barcode transcript

Life of this patent

Encoding of dna vector identity via iterative hybridization detection of a barcode transcript

Life of this patent

Abstract

Claims36