# US 12,180,486 B2 · Compositions of self-reporting transposon (SRT) constructs and methods for mapping transposon insertions

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- Number: US 12,180,486 B2
- Document ID: US12180486B2
- Status: Granted
- Kind: B2
- Application number: 16/711,011
- Filed: 2019-12-11
- Published: 2020-06-11
- Granted: 2024-12-31
- Earliest claimed: 2018-12-11
- Assignees: Washington University
- Applicants: Robi D. Mitra; Arnav Moudgil; Michael Nathaniel Wilkinson; Zongtai Qi
- Inventors: Robi D. Mitra; Arnav Moudgil; Michael Nathaniel Wilkinson; Zongtai Qi
- Web page: https://patentgrep.com/patent/US12180486B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/907: {in mammalian cells}
- C12N 2310/11: Antisense
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3513: Protein; Peptide
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Among the various aspects of the present disclosure is the provision of compositions and methods for mapping transposon insertions. Applications can include mapping the locations of self-reporting transposons (SRTs) from thousands of single cells in parallel, while simultaneously measuring mRNA abundance from the same single cells; analyzing genome-associated protein (GAP) (e.g., transcription factor) binding/interactions in a small number of cells in bulk, without single cell resolution; lineage tracing; or as an improved readout for transposon mutagenesis screens.

## Claims

### Claim 1

A self-reporting transposon (SRT) construct comprised of a transposon containing transposon DNA and at least one promoter; wherein the promoter is capable of driving transcription of DNA through at least one transposon end after the SRT construct is inserted into genomic DNA so that a portion of the transposon DNA, the at least one transposon end, and genomic DNA flanking the transposon end is transcribed into RNA; and wherein the transposon does not contain a poly-adenylation (poly-A) termination signal in the transcribed region.

### Claim 2 (depends on 1)

The SRT construct of claim 1, wherein the promoter is an inducible promoter.

### Claim 3 (depends on 2)

The SRT construct of claim 2, wherein the inducible promoter is capable of being induced by a chemical inducer and light.

### Claim 4 (depends on 1)

The SRT construct of claim 1, wherein the promoter comprises an EF1α promoter, a CAG promoter, a PGK promoter, a Tet-on or Tet-off promoter, a T7 promoter, or a CMV promoter.

### Claim 5 (depends on 1)

The SRT construct of claim 1, wherein the promoter drives expression of a reporter gene incorporated in the transposon.

### Claim 6 (depends on 5)

The SRT construct of claim 5, wherein the reporter gene is selected from the group consisting of:
a gene encoding a fluorescent protein;
a gene capable of use as a selectable marker by conferring resistance to a chemical agent that kills eukaryotic or prokaryotic cells; and
a gene encoding an enzyme capable of converting a chemical substrate into a colorimetric reporter, a luminescent reporter, or a fluorescent reporter.

### Claim 7 (depends on 6)

The SRT construct of claim 6, wherein the gene encoding a fluorescent protein comprises green fluorescent protein, tdTomato, eGFP, or eCFP.

### Claim 8 (depends on 6)

The SRT construct of claim 6, wherein the gene capable of use as a selectable marker comprises puromycin N-acetyl-transferase, an aminoglycoside 3′ phosphotransferase gene encoded by Tn5 and Tn601, or hygromycin phosphotransferase.

### Claim 9 (depends on 1)

The SRT construct of claim 1, wherein the transposon encodes a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).