# US 2025/0084484 A1 · Methods and compositions for transcriptome analysis

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- Number: US 2025/0084484 A1
- Document ID: US20250084484A1
- Status: Published application
- Kind: A1
- Application number: 18/728,252
- Filed: 2023-01-10
- Published: 2025-03-13
- Earliest claimed: 2022-01-12
- Assignees: Jumpcode Genomics, Inc.
- Applicants: Jumpcode Genomics, Inc.
- Inventors: Keith BROWN
- Web page: https://patentgrep.com/patent/US20250084484A1

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6855: Ligating adaptors
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12Q 2600/158: Expression markers
- 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

Provided herein are methods and compositions for analysis of a transcriptome, for example detection of a RNA isoform, of a cell, tissue, organ, or subject.

## Claims

### Claim 1

A method of detecting an RNA isoform comprising:
(a) contacting a plurality of RNA molecules to a oligo(dT) primer and a reverse transcriptase to produce a plurality of cDNA molecules;
(b) attaching adaptors to a 5′ end and a 3′ end of each of said plurality of cDNA molecules;
(c) amplifying said plurality of cDNA molecules with a first primer and a second primer that bind to said adaptors to produce a plurality of amplification products, wherein said first primer or said second primer comprises a detectable label;
(d) capturing at least a portion of said plurality of amplification products using an agent that binds to said detectable label;
(e) contacting at least a portion of said plurality of amplification products to a guide RNA directed endonuclease and at least one guide RNA that specifically binds to a 5′ end or a 3′ end of at least a subset of said plurality of amplification products, thereby cleaving a 5′ end or a 3′ end of said plurality of amplification products to produce a plurality of cleaved amplification products; and
(f) isolating said plurality of cleaved amplification products, thereby detecting said RNA isoform.

### Claim 2 (depends on 1)

The method of claim 1, wherein said attaching of (b) comprises ligating.

### Claim 3 (depends on 1)

The method of claim 1, wherein said attaching of (b) occurs concurrently with (a) wherein said oligo(dT) primer comprises said adaptor.

### Claim 4 (depends on 1, 2, 3)

The method of any one of claims 1 to 3, wherein said first primer binds to said adaptor at said 5′ end of each of said plurality of cDNA molecules and said second primer binds to said adaptor at said 3′ end of each of said plurality of cDNA molecules.

### Claim 5 (depends on 4)

The method of claim 4, wherein said first primer comprises said detectable label and said guide RNA binds to said 5′ end of said subset of said plurality of amplification products.

### Claim 6 (depends on 4)

The method of claim 4, wherein said second primer comprises said detectable label and said guide RNA binds to said 3′ end of said subset of said plurality of amplification products.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1 to 6, further comprising subsequent to (a) dividing said plurality of cDNA molecules to at least a first reaction volume and a second reaction volume.

### Claim 8 (depends on 7)

The method of claim 7, wherein in said first reaction volume said first primer comprises said detectable label and said guide RNA binds to said 5′ end of said plurality of amplification products.

### Claim 9 (depends on 7, 8)

The method of claim 7 or claim 8, wherein in said second reaction volume said second primer comprises said detectable label and said guide RNA binds to said 3′ end of said plurality of amplification products.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1 to 9, wherein said detectable label comprises biotin.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1 to 10, wherein said agent comprises streptavidin.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any one of claims 1 to 11, wherein said at least one guide RNA binds specifically to a 5′ end or a 3′ end of said RNA isoform.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method of any one of claims 1 to 12, wherein said at least one guide RNA is provided in an amount relative to an expected amount of said RNA isoform.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method of any one of claims 1 to 13, further comprising sequencing said plurality of cleaved amplification products.

### Claim 15

A method of detecting an RNA isoform comprising:
(a) contacting a plurality of RNA molecules to a oligo(dT) primer and a reverse transcriptase to produce a plurality of cDNA molecules;
(b) attaching adaptors to a 5′ end and a 3′ end of each of said plurality of cDNA molecules;
(c) amplifying said plurality of cDNA molecules with a first primer and a second primer that bind to said adaptors to produce a plurality of amplification products;
(d) contacting at least a portion of said plurality of amplification products to a cleavage deficient guide RNA directed endonuclease and at least one guide RNA that specifically binds to a 5′ end or a 3′ end of at least a subset of said plurality of amplification products;
(e) capturing at least a portion of said plurality of amplification products using an agent that binds to said cleavage deficient guide RNA directed endonuclease, thereby detecting said RNA isoform.

### Claim 16 (depends on 15)

The method of claim 15, wherein said attaching of (b) comprises ligating.

### Claim 17 (depends on 15)

The method of claim 15, wherein said attaching of (b) occurs concurrently with (a) wherein said oligo(dT) primer comprises said adaptor.

### Claim 18 (depends on 15, 16, 17)

The method of any one of claims 15 to 17, wherein said at least one guide RNA binds specifically to a 5′ end or a 3′ end of said RNA isoform.

### Claim 19 (depends on 15, 16, 17)

The method of any one of claims 15 to 17, wherein said at least one guide RNA is provided in an amount relative to an expected amount of said RNA isoform.

### Claim 20 (depends on 15, 16, 17, 18, 19)

The method of any one of claims 15 to 19, further comprising sequencing said plurality of captured amplification products.