# US 2021/0340527 A1 · Encoding of dna vector identity via iterative hybridization detection of a barcode transcript

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0340527 A1
- Document ID: US20210340527A1
- Status: Published application
- Kind: A1
- Application number: 17/321,282
- Filed: 2021-05-14
- Published: 2021-11-04
- Earliest claimed: 2015-03-16
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Inventors: Paul Blainey; David Feldman
- Web page: https://patentgrep.com/patent/US20210340527A1

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim at position 1

1-36. (canceled)

### Claim 37

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.

### Claim 38 (depends on 37)

The method of claim 37, further comprising determining an observed phenotype for each cell or cell population by capturing a microscopic image of the cell or cell population; and correlating the observed phenotype to the one or more detected optical barcodes.

### Claim 39 (depends on 37)

The method of claim 37, wherein detecting the one or more optical barcodes comprises:
delivering a probe set to the cell or cell population, each probe in the probe set comprising a sequence that hybridizes to one of the possible nucleic acid sequences at the first segment of the optical barcode on the RNA, wherein different probe sequences are labeled with different optically detectable labels such that each nucleic acid sequence at the first segment of the optical barcode is labeled with a different optically detectable label;
determining the nucleotide sequence at the first segment of each barcode by detecting the optically detectable labels; and
repeating the delivering and determining steps for each segment in the barcode to detect all remaining segments in the optical barcode, wherein the order in which the probes in the probe set bind to the segments of the optical barcode identify each optical barcode and thereby identify the one or more genetic perturbations introduced into each cell or cell population.

### Claim 40 (depends on 38)

The method of claim 38, further comprising generating a cDNA copy of the RNA prior to detecting the optical barcode.

### Claim 41 (depends on 40)

The method of claim 40, further comprising amplifying the generated cDNA copy prior to detecting the optical barcode.

### Claim 42 (depends on 37)

The method of claim 37, wherein detecting the optical barcode comprises detecting the nucleic acid sequence of the ordered segments in the optical barcode using an in situ sequencing method.

### Claim 43 (depends on 42)

The method of claim 42, wherein the in situ sequencing method is fluorescent in situ RNA sequencing (FISSEQ) or in situ mRNA-seq.

### Claim 44 (depends on 37)

The method of claim 37, wherein detecting the one or more optical barcodes comprises:
delivering an intermediate probe set to the cell or cell population, each probe in the probe set comprising a sequence that hybridizes to one of the possible nucleic acid sequences at the first segment of the optical barcode on the RNA;
delivering a second probe set that comprises probes that bind to the intermediate probes, the probes in the second probe set comprising an optically detectable label;
determining the nucleotide sequence at the first segment of each barcode by detecting the optically detectable labels; and
repeating the delivering and determining steps for each segment in the barcode to detect all remaining segments in the optical barcode, wherein the order in which the probes in the probe set bind to the segments of the optical barcode identify each optical barcode and thereby identify the one or more genetic perturbations introduced into each cell or cell population.

### Claim 45 (depends on 44)

The method of claim 44, wherein the intermediate probes are branched probes that facilitate the binding of multiple probes from the second probe set to a single intermediate probe.

### Claim 46 (depends on 37)

The method of claim 37, wherein the RNA comprising the barcode further comprises a localization signal localizing the RNA comprising the barcode to a specific location within the cell.

### Claim 47 (depends on 46)

The method of claim 46, wherein the cell localization signal is a nuclear localization signal.

### Claim 48 (depends on 37)

The method of claim 37, wherein the vector further comprises nucleic acid sequences further encoding the site-specific nuclease.

### Claim 49 (depends on 48)

The method of claim 48, wherein the genetic perturbation is a guide RNA.

### Claim 50 (depends on 37)

The method of claim 37, wherein the vector encodes guide RNA and the site-specific nuclease is an RNA-guided DNA endonuclease.

### Claim 51 (depends on 5050)

The method of claim 5050, wherein the RNA-guided DNA endonuclease is dCas9.

### Claim 52 (depends on 51)

The method of claim 51, wherein the dCas9 is fused to a second domain.

### Claim 53 (depends on 52)

The method of claim 52, wherein the second domain is a nickase.

### Claim 54 (depends on 53)

The method of claim 53, wherein the nickase is Fok1.

### Claim 55 (depends on 52)

The method of claim 52, wherein the second domain is selected from a transcriptional activator, or a transcriptional repressor, a recombinase, a transposase, a DNA methyltransferase or a histone methyltransferase.

### Claim 56 (depends on 50)

The method of claim 50, further comprising generating, prior to the introducing, a set of gateway vectors, the gateway vector comprising a guide RNA and corresponding optical barcode assigned to each guide RNA such that the guide RNA and optical barcode are adjacent to one another; and identifying each guide RNA and optical barcode pair using short-read DNA sequencing.

### Claim 57 (depends on 37)

The method of claim 37, wherein the optical barcode further comprises a unique molecular identifier (UMI), and each guide RNA sequence comprises a barcode-UMI specific homology sequence to facilitate one to one cloning of guide RNAs to corresponding optical barcodes.

### Claim 58 (depends on 37)

The method of claim 37, wherein the site-specific nuclease is a zinc-finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

### Claim 59 (depends on 37)

The method of claim 37, wherein the one or more genetic perturbations comprises one or more nucleotide insertions, deletions, or substitutions.

### Claim 60 (depends on 37)

The method of claim 37, wherein the RNA comprising the barcode further comprises a premature termination signal to prevent translation of the RNA comprising the barcode.

### Claim 61 (depends on 37)

The method of claim 37, wherein each nucleic acid sequence at each segment in the barcode is between approximately 100 to approximately 200 nucleotides.

### Claim 62 (depends on 37)

The method of claim 37, wherein the barcode comprises 3 to 8 segments.

### Claim 63 (depends on 39)

The method of claim 39, wherein each probe set comprises 3, 4, or 5 distinct optically detectable labels.

### Claim 64 (depends on 37)

The method of claim 37, wherein the optically detectable label is a fluorophore.

### Claim 65 (depends on 37)

The method of claim 37, wherein the optically detectable label is a quantum dot.

### Claim 66 (depends on 37)

The method of claim 37, wherein the optically detectable label is an object of a particular size, shape, color, or combination thereof.

### Claim 67 (depends on 37)

The method of claim 37, further comprising sequencing the one or more vectors to identify the sequence defining the barcode associated with the one or more genetic perturbations.

### Claim 68 (depends on 37)

The method of claim 37, wherein the optical barcode further comprises a unique molecular identifier (UMI).

### Claim 69 (depends on 68)

The method of claim 68, further comprising sequencing the UMI and optical barcode to identify the UMI assigned to each optical barcode thereby allowing short sequencing of the UMI to identify the optical barcode associated with each vector and genetic perturbation or guide RNA encoded by that vector.

### Claim 70 (depends on 37)

The method of claim 37, further comprising selecting cells with increased RNA expression from the one or more vectors introduced into the cells, wherein the one or more vectors further encode a degradation domain fused to an antibiotic resistance protein such that rapid degradation of the expressed antibiotic resistance protein can be induced such that only cells with increased RNA expression are selected in the presence of an antibiotic.

### Claim 71 (depends on 37)

The method of claim 37, wherein the one or more genetic perturbations is an RNAi.