Trans-splicing transcriptome profiling
| Title: | Trans-splicing transcriptome profiling |
|---|---|
| Patent Number: | 9,752,179 |
| Publication Date: | September 05, 2017 |
| Appl. No: | 14/208446 |
| Application Filed: | March 13, 2014 |
| Abstract: | The present invention provides a method of identifying mRNA transcripts in the transcriptome of a cell comprising i) delivering into the cell a donor expression vector comprising nucleotides in a sequence encoding a trans-splicing barcode cassette, ii) exposing the cell to conditions such that the cell produces multiple copies of the trans-splicing barcode cassette encoded by the donor expression vector, which multiple copies of the trans-splicing barcode cassette each splice the barcode polynucleotide onto a mRNA transcript of the cell, thereby forming multiple mRNA transcripts of the cell, each spliced to the barcode polynucleotide; and iii) identifying the multiple mRNA transcripts that are spliced to the barcode polynucleotides, thereby identifying mRNA transcripts in the transcriptome of the cell. |
| Inventors: | Zador, Anthony M. (Cold Spring Harbor, NY, US); Peikon, Ian D. (Cold Spring Harbor, NY, US); Znamenskiy, Petr (Basel, CH) |
| Assignees: | COLD SPRING HARBOR LABORATORY (Cold Spring Harbor, NY, US) |
| Claim: | 1. A method of identifying mRNA transcripts in the transcriptomes of multiple cells within a population of cells comprising i) delivering into each of at least two cells a donor expression vector comprising nucleotides in a sequence encoding a trans-splicing barcode cassette having the same nucleotide sequence, wherein the trans-splicing barcode cassette comprises a) a first portion, the nucleotide sequence of which encodes an intron comprising as part of its 3′ end, or followed at its 3′ end by a splice-site nucleotide sequence; followed at its 3′ end by, b) a second portion, the nucleotide sequence of which encodes a barcode polynucleotide; followed at its 3′ end by c) a third portion, which encodes a nucleotide identification element sequence, wherein the portion of the nucleotide sequence of the expression vector that encodes the barcode polynucleotide is altered within the at least two cells, such that each of the at least two cells within the population expresses copies of the trans-splicing barcode cassette having a unique barcode polynucleotide sequence, ii) exposing the cells to conditions such that each of the at least two cells produces multiple copies of the trans-splicing barcode cassette encoded by the donor expression vector, which multiple copies of the trans-splicing barcode cassette each splice the barcode polynucleotide onto a mRNA transcript of the at least two cells, thereby forming multiple spliced mRNA transcripts of the cell, each spliced to the barcode polynucleotide; and iii) identifying the multiple mRNA transcripts that are spliced to the barcode polynucleotides, thereby identifying mRNA transcripts in the transcriptomes of multiple cells within a population of cells. |
| Claim: | 2. The method of claim 1 , wherein the intron comprises the Adeno Virus immediate early intron. |
| Claim: | 3. The method of claim 2 , wherein the Adeno Virus immediate early intron is followed at its 3′ end by the splice-site nucleotide sequence. |
| Claim: | 4. The method of claim 1 , wherein the splice-site nucleotide sequence is CAG. |
| Claim: | 5. The method of claim 1 , wherein the intron is other than the Adeno Virus immediate early intron. |
| Claim: | 6. The method of claim 1 , wherein the population of cells is an in vitro culture of cells. |
| Claim: | 7. The method of claim 1 , wherein the population of cells is within the tissue obtained from or present within an organism. |
| Claim: | 8. The method of claim 1 , wherein a recombinase alters the nucleotide sequence of the expression vector that encodes the barcode nucleotide sequence. |
| Claim: | 9. The method of claim 8 , wherein the recombinase is expressed in each cell into which a trans-splicing barcode cassette is delivered. |
| Claim: | 10. The method of claim 1 , wherein the barcode polynucleotide comprises multiple unique DNA segments of multiple nucleotides separated by recombination sites, such that the multiple unique DNA segments are recombined by a recombinase when the recombinase is co-expressed with the donor expression vector in each of the at least two cells. |
| Claim: | 11. The method of claim 10 , wherein the barcode polynucleotide sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 unique DNA segments. |
| Claim: | 12. The method of claim 11 , wherein each unique DNA segment comprises 1-200 nucleotides. |
| Claim: | 13. The method of claim 10 , wherein the recombinase is Rci, and the recombination sites are each a pair of sfx recombination sites. |
| Claim: | 14. The method of claim 13 , wherein the sfx recombination sites within each pair of sfx recombination sites are in an opposing orientation to each other. |
| Claim: | 15. The method of claim 10 , wherein the recombinase is Cre, and the recombination sites are loxP sites. |
| Claim: | 16. The method of claim 10 , wherein the recombinase is Flp, and the recombination sites are FRT sites. |
| Claim: | 17. The method of claim 10 , wherein the recombinase is PhiC31, and the recombination sites are att sites. |
| Claim: | 18. The method of claim 1 , wherein in step iii) the nucleotide identification element sequence is used to identify the multiple mRNA transcripts that are spliced to the barcode polynucleotide; or wherein the nucleotide identification element sequence comprises a nucleotide sequence which is used to a) identify the multiple mRNA transcripts that are spliced to the barcode polynucleotides, b) reverse transcribe the barcode polynucleotide and at least a portion of the multiple mRNA transcripts that are spliced to the barcode polynucleotide, and/or c) sequence the barcode polynucleotide and at least a portion of the multiple mRNA transcripts that are spliced to the barcode polynucleotide. |
| Claim: | 19. The method of claim 1 , wherein each donor expression vector further comprises nucleotides in a sequence encoding the recombinase, or wherein a second expression vector comprising nucleotides in a sequence encoding the recombinase is delivered into the at least two cells concurrently with the donor expression vector. |
| Claim: | 20. A method of identifying mRNA transcripts in the transcriptome of a cell comprising i) delivering into the cell a donor expression vector comprising nucleotides in a sequence encoding a trans-splicing barcode cassette, wherein the trans-splicing barcode cassette comprises a) a first portion, the nucleotide sequence of which encodes an intron comprising as part of its 3′ end, or followed at its 3′ end by a splice-site nucleotide sequence; followed at its 3′ end by, b) a second portion, the nucleotide sequence of which encodes a barcode polynucleotide, which is alterable by a recombinase; followed at its 3′ end by c) a third portion, which encodes a nucleotide identification element sequence, wherein a recombinase alters the nucleotide sequence of the expression vector that encodes the barcode nucleotide sequence, ii) exposing the cell to conditions such that the cell produces multiple copies of the trans-splicing barcode cassette encoded by the donor expression vector, which multiple copies of the trans-splicing barcode cassette each splice the barcode polynucleotide onto a mRNA transcript of the cell, thereby forming multiple mRNA transcripts of the cell, each spliced to the barcode polynucleotide; and iii) identifying the multiple mRNA transcripts that are spliced to the barcode polynucleotides, thereby identifying mRNA transcripts in the transcriptome of the cell. |
| Claim: | 21. The method of claim 20 , wherein in step iii) the nucleotide identification element sequence is used to identify the multiple mRNA transcripts that are spliced to the barcode polynucleotide; or wherein the nucleotide identification element sequence comprises a nucleotide sequence which is used to a) identify the multiple mRNA transcripts that are spliced to the barcode polynucleotide, b) reverse transcribe the barcode polynucleotide and at least a portion of the multiple mRNA transcripts that are spliced to the barcode polynucleotide, and/or c) sequence the barcode polynucleotide and at least a portion of the multiple mRNA transcripts that are spliced to the barcode polynucleotide. |
| Claim: | 22. The method of claim 20 , a) wherein the recombinase is expressed in the cell into which the donor expression vector is delivered; b) wherein the donor expression vector further comprises nucleotides in a sequence encoding the recombinase; or c) wherein a second expression vector comprising nucleotides in a sequence encoding the recombinase is delivered into the cell concurrently with the donor expression vector. |
| Claim: | 23. The method of claim 20 , wherein the barcode polynucleotide comprises multiple unique DNA segments of multiple nucleotides separated by recombination sites, such that the multiple unique DNA segments are recombined by the recombinase when the recombinase is co-expressed with the donor expression vector in the cell. |
| Claim: | 24. The method of claim 23 , a) wherein the recombinase is Rci, and the recombination sites are each a pair of sfx recombination sites; b) wherein the recombinase is Rci, and the recombination sites are each a pair of sfx recombination sites, and wherein the sfx recombination sites within each pair of sfx recombination sites are in an opposing orientation to each other; c) wherein the recombinase is Cre, and the recombination sites are loxP sites; d) wherein the recombinase is Flp, and the recombination sites are FRT sites; or e) wherein the recombinase is PhiC31, and the recombination sites are attB and attP sites. |
| Patent References Cited: | 8003381 August 2011 Klimyuk et al. 9447411 September 2016 Chenchik 2012/0245039 September 2012 Roth 2014/0283156 September 2014 Zador et al. |
| Other References: | Kohler et al. (1999) Trans-splicing Ribozymes for Targeted Gene Delivery, J. Mol. Biol. 285:1935-1950. cited by applicant Zador et al. (2012) Sequencing the Connectome, PLoS Biology 10(10):e1001411. cited by applicant |
| Primary Examiner: | Woolwine, Samuel |
| Attorney, Agent or Firm: | Gershik, Gary J. Cooper & Dunham LLP |
| Accession Number: | edspgr.09752179 |
| Database: | USPTO Patent Grants |
| Language: | English |
|---|