# US 2021/0340566 A1 · Compositions and methods for differential cas9 gene labeling and/or editing

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- Number: US 2021/0340566 A1
- Document ID: US20210340566A1
- Status: Published application
- Kind: A1
- Application number: 17/200,398
- Filed: 2021-03-12
- Published: 2021-11-04
- Earliest claimed: 2013-12-17
- Assignees: University of Massachusetts; University of Central Florida Research Foundation, Inc.
- Applicants: University of Massachusetts; University of Central Florida Research Foundation, Inc.
- Inventors: THORU PEDERSON; SCOT ANDREW WOLFE; HANHUI MA; Metewo Selase Kosi Enuameh; Nicola Anne Kearns; Ryan Michael Jude Genga; Rene Maehr; Shaojie Zhang; Ardalan Naseri; Manuel Garber
- Web page: https://patentgrep.com/patent/US20210340566A1

## CPC

- C12N 15/86: Viral vectors
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/907: {in mammalian cells}
- C12Q 1/6841: In situ hybridisation
- C12N 2501/65: MicroRNA
- C12N 2501/70: Enzymes
- C12N 2510/00: Genetically modified cells
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present disclosure relates to methods of and systems for modifying the transcriptional regulation of stem or progenitor cells to promote their differentiation or reprogramming of somatic cells. Further, the labeling and editing of human genomic loci in live cells with three orthogonal CRISPR/Cas9 components allow multicolor detection of genomic loci with high spatial resolution, which provides an avenue for barcoding elements of the human genome in the living state.

## Claims

### Claim at position 1

1-20. (canceled)

### Claim 21

A method, comprising:
a) providing;
i) a live cell comprising a first genomic locus and a second genomic locus and a catalytically active Cas9;
ii) a first deoxyribonucleic acid (DNA) construct encoding a first catalytically inactive Cas9 (dCas) protein and a first fluorescent label protein having a first color, wherein said first dCas protein binds to a first single guide ribonucleic acid (sgRNA) that comprises a first complementary sequence to said first genomic locus;
iii) a second DNA construct encoding a second dCas and a second fluorescent label protein having a second color, wherein said second dCas protein binds to a second sgRNA that comprises a second complementary sequence to said second genomic locus;
b) transfecting said live cell with said first DNA construct, said second DNA construct, said first sgRNA and said second sgRNA, wherein said first dCas construct and said second dCas construct are expressed;
c) detecting said first color in said first genomic locus or said second color in said second genomic locus;
d) determining that said catalytically active Cas9 has edited: i) only said first genomic locus when only said first color is detected; ii) both said first genomic locus and said second genomic locus when both said first color and said second color is detected; or iii) only said second genomic locus with only said second color is detected.

### Claim 22 (depends on 21)

The method of claim 21, wherein said detecting is performed by fluorescence microscopy.

### Claim 23 (depends on 21)

The method of claim 21, wherein said detecting is performed by fluorescence activated cell sorting.

### Claim 24 (depends on 21)

The method of claim 21, wherein said detecting is performed without a method selected from the group consisting of nucleic acid sequencing, reverse transcriptase polymerase chain reaction and gel electrophoresis.

### Claim 25 (depends on 21)

The method of claim 21, wherein said first color and said second color are different.

### Claim 26 (depends on 21)

The method of claim 21, wherein said first DNA construct fluffier comprises a first DNA binding protein.

### Claim 27 (depends on 21)

The method of claim 21, wherein said second DNA construct further comprises a second DNA binding protein.

### Claim 28 (depends on 21)

The method of claim 21, wherein said first dCas9 protein and said second dCas9 protein are orthogonal.

### Claim 29 (depends on 21)

The method of claim 21, wherein said first dCas9 protein and said second dCas9 protein are derived from a bacterial species selected from the group consisting of S. pyogenes, N. meningitidis and S. thermophilus.

### Claim 30

A method, comprising:
a) providing;
i) a live cell comprising a genomic locus;
ii) a first deoxyribonucleic acid (DNA) construct encoding a catalytically active Cas9 protein that binds to a full length single guide ribonucleic acid that comprises a first complementary sequence to said genomic locus;
iii) a second DNA construct encoding a second catalytically active Cas9 protein and a fluorescent label protein having a color, wherein said second catalytically active Cas9 protein binds to a truncated sgRNA that comprises a second complementary sequence to said genomic locus;
b) transfecting said live cell with said first DNA construct, said second DNA construct, said full length sgRNA and said truncated sgRNA, wherein said first dCas construct and said second dCas construct are expressed;
c) detecting said color in said genomic locus;
d) determining that said catalytically active Cas9 has edited said genomic locus based on said detected color.

### Claim 31 (depends on 30)

The method of claim 30, wherein said detecting is performed by fluorescence microscopy.

### Claim 32 (depends on 30)

The method of claim 30, wherein said detecting is performed by fluorescence activated cell sorting.

### Claim 33 (depends on 30)

The method of claim 30, wherein said detecting is performed without a method selected from the group consisting of nucleic acid sequencing, reverse transcriptase polymerase chain reaction and gel electrophoresis.

### Claim 34 (depends on 30)

The method of claim 30, wherein said color is selected from the group consisting of red, green and blue.

### Claim 35 (depends on 30)

The method of claim 30, wherein said catalytically active Cas9 protein is derived from a bacterial species selected from the group consisting of S. pyogenes, N. meningitidis and S. thermophilus.