Pooled method for high throughput screening of trans factors affecting rna levels

Patent metadata

Field	Value
Publication number	US-2016362684-A1
Application number	US-201615175977-A
Country	US
Kind code	A1
Filing date	Jun 7, 2016
Priority date	Dec 18, 2013
Publication date	Dec 15, 2016
Grant date	—

Abstract

Provided herein are methods directed to multiplexed detection of the modulation of transcriptional activity using perturbation elements.

First claim

1 . A method of multiplexed detection of modulation of transcriptional activity caused by a plurality of perturbation elements, said method comprising:
(i) introducing a plurality of delivery vectors into a cell culture, wherein each delivery vector within said plurality of delivery vectors individually comprises:
(a) a perturbation element identifying DNA sequence operatively linked to a promoter of interest; and
(b) a perturbation element-encoding DNA sequence operatively linked to a constitutive promoter; and
wherein said cells of said cell culture integrate one delivery vector per cell; (ii) allowing said cell culture to express said plurality of delivery vectors, wherein said expression forms a plurality of perturbation element identifying RNA sequences from said perturbation element identifying DNA sequences and a plurality of perturbation elements from said perturbation element-encoding DNA sequences; (iii) allowing each perturbation element within said plurality of perturbation elements to modulate transcriptional activity of each corresponding promoter of interest; (iv) detecting an amount of each perturbation element identifying RNA sequence within said plurality of perturbation element identifying RNA sequences; (v) comparing said amount of each perturbation element identifying RNA sequence within said plurality of perturbation element identifying RNA sequences to an amount of each corresponding perturbation element identifying DNA sequences, thereby detecting modulation of transcriptional activity caused by each of said plurality of perturbation elements.

2 . The method of claim 1 , wherein said perturbation element identifying DNA sequence is about 15 nucleotides to about 500 nucleotides in length.

3 . The method of claim 2 , wherein said perturbation element identifying DNA sequence is about 15 to about 100 nucleotides in length.

4 . The method of claim 2 , wherein said perturbation element identifying DNA sequence is about 20 nucleotides in length.

5 . The method of claim 4 , wherein each delivery vector within said plurality of delivery vectors further comprises an auxiliary-DNA sequence operatively linked to said perturbation element identifying DNA sequence.

6 . The method of claim 5 , wherein said auxiliary-DNA sequence is operatively linked to a 5′ end of said perturbation element identifying DNA sequence.

7 . The method of claim 6 , wherein said perturbation element identifying DNA sequence is located in the 3′-UTR of said auxiliary-DNA sequence.

8 . The method of claim 1 , wherein said plurality of delivery vectors are viral plasmids, 2-micron plasmids, or CEN/ARS plasmids.

9 . The method of claim 8 , wherein said plurality of delivery vectors are viral plasmids.

10 . The method of claim 9 , wherein said plurality of delivery vectors are lentiviral delivery vectors.

11 . The method of claim 1 , wherein said promoter of interest is operatively linked to a 5′ end of said perturbation element identifying DNA sequence, wherein said constitutive promoter is linked to a 3′ end of said perturbation element identifying DNA sequence, and wherein said perturbation element-encoding DNA sequence is operatively linked to a 3′ end of said constitutive promoter.

12 . The method of claim 1 , wherein said plurality of perturbation elements comprises shRNA, miRNA, siRNA or CRISPR guide RNA.

13 . The method of claim 1 , wherein said plurality of perturbation elements comprises a protein-perturbation element.

14 . The method of claim 1 , wherein said detecting an amount of each perturbation element identifying RNA sequence within said plurality of perturbation element identifying RNA sequences comprises sequencing said plurality of perturbation element identifying RNA sequences and counting the frequency of each perturbation element identifying RNA sequence within said plurality of perturbation element identifying RNA sequences.

15 . The method of claim 14 , wherein an increased count of a perturbation element identifying RNA sequence relative to an amount of its corresponding perturbation element identifying DNA sequence indicates increased transcriptional activity.

16 . The method of claim 1 , wherein said cell is a terminally differentiated cell.

17 . A method of multiplexed quantitative detection of modulation of transcriptional activity caused by a plurality of perturbation elements, said method comprising:
(i) introducing a plurality of delivery vectors into a cell culture, wherein each delivery vector within said plurality of delivery vectors individually comprises:
(a) a perturbation element identifying DNA sequence operatively linked to a promoter of interest; and
(b) a RNA-perturbation element-encoding DNA sequence operatively linked to a constitutive promoter; and
wherein said cells of said cell culture integrate one delivery vector per cell; (ii) allowing said cell culture to express said plurality of delivery vectors, wherein said expression forms a plurality of perturbation element identifying RNA sequences from said perturbation element identifying DNA sequences and a plurality of RNA-perturbation elements from said perturbation element-encoding DNA sequences; (iii) allowing each RNA-perturbation element within said plurality of perturbation elements to modulate transcriptional activity of each corresponding promoter of interest; (iv) lysing the cells in the cell culture and extracting nucleic acids; (v) sequencing each perturbation element identifying RNA sequence within said plurality of perturbation element identifying RNA sequences, thereby determining an amount of each perturbation element identifying RNA sequence; (v) comparing said amount of each perturbation element identifying RNA sequence to an amount of each corresponding perturbation element identifying DNA sequences, thereby quantitatively detecting modulation of transcriptional activity caused by each of said plurality of perturbation elements.

18 . (canceled)

19 . A kit for multiplexed quantitative detection of modulation of transcriptional activity caused by a plurality of perturbation elements, said kit comprising:
(i) a plurality of delivery vectors, wherein each delivery vector within said plurality of delivery vectors individually comprises:
(a) a perturbation element identifying DNA sequence operatively linked to a promoter of interest; and
(b) a perturbation element-encoding DNA sequence operatively linked to a constitutive promoter;
(ii) a first oligonucleotide primer substantially complementary to and hybridizable with said perturbation element identifying DNA sequence; and (iii) a second oligonucleotide primer substantially complementary to and hybridizable with a perturbation element identifying RNA sequence expressed from said perturbation element identifying DNA sequence.

20 . The kit of claim 19 further comprising a third oligonucleotide primer substantially complementary to and hybridizable with said perturbation element-encoding DNA sequence.

Assignees

• Univ California

Inventors

• Brandman Onn
• Weissman Jonathan
• Jan Calvin H
• Gilbert Luke A

Classifications

• C12N15/1034 Primary

  Isolating an individual clone by screening libraries · CPC title

• C12N15/1086 Primary

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title

• B01J2219/00547

  Bar codes · CPC title

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• B01J2219/00743

  Cells · CPC title