MULTIPLEX HOMOLOGY-DIRECTED REPAIR

• US 20160076093A1
• Filed: 08/04/2015
• Published: 03/17/2016
• Est. Priority Date: 08/04/2014
• Status: Abandoned Application

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Boyle, Evan, Findlay, Gregory, Shendure, Jay
• Application Number:
  US14/818,217
• Publication Number:
  US 20160076093A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

Disclosed is a method for introducing a plurality of programmed nucleotide modifications into a single locus of a desired genomic DNA sequence in a single experiment. The method entails synthesizing a homology-directed repair (HDR) library comprising a plurality of oligonucleotides, wherein each oligonucleotide comprises a programmed nucleotide modification in the locus of the desired genome, and co-transfecting a population of cells with (i) an expression system capable of expressing Cas9 and a single guide RNA (sgRNA) and (ii) introducing a plurality of programmed nucleotide modifications to the locus of the desired genomic DNA sequence in one or more cells of the population. Also disclosed are methods for analyzing the functional consequence of a genomic mutation and for genomic screening.

Claims

1. A method for introducing a plurality of programmed nucleotide modifications into a single locus of a desired genomic DNA sequence, comprising:

(a) synthesizing a homology-directed repair (HDR) library comprising a plurality of oligonucleotides, wherein each oligonucleotide comprises a programmed nucleotide modification in the locus of the desired genome; and

(b) co-transfecting a population of cells with (i) an expression system capable of expressing Cas9 and a single guide RNA (sgRNA), and (ii) the HDR library, wherein the expression system is capable of introducing the plurality of oligonucleotides having the programmed nucleotide modifications to the locus of the desired genomic DNA sequence in one or more cells of the population;

wherein the method is carried out in a single experiment.

2. The method of claim 1, wherein each programmed nucleotide modification is a single nucleotide variant.

3. The method of claim 1, wherein the HDR library is constructed using an oligonucleotide comprising a degenerate sequence.

4. The method of claim 3, wherein the degenerate sequence is between 1 and 100 nucleotides in length.

5. The method of claim 4, wherein the HDR library comprises a set of oligonucleotides having at least 100 unique programmed nucleotide modifications, a least 200 unique programmed nucleotide modifications, at least 300 unique programmed nucleotide modifications, at least 400 unique programmed nucleotide modifications, at least 500 unique programmed nucleotide modifications, at least 600 unique programmed nucleotide modifications, at least 700 unique programmed nucleotide modifications, at least 800 unique programmed nucleotide modifications, at least 900 unique programmed nucleotide modifications, at least 1,000 unique programmed nucleotide modifications, at least 3,000 unique programmed nucleotide modifications, at least 4,000 unique programmed nucleotide modifications, at least 5,000 unique programmed nucleotide modifications, at least 6,000 unique programmed nucleotide modifications, at least 7,000 unique programmed nucleotide modifications, at least 8,000 unique programmed nucleotide modifications, at least 9,000 unique programmed nucleotide modifications, at least 10,000 unique programmed nucleotide modifications, at least 12,000 unique programmed nucleotide modifications, at least 14,000 unique programmed nucleotide modifications, at least 16,000 unique programmed nucleotide modifications, at least 18,000 unique programmed nucleotide modifications, at least 20,000 unique programmed nucleotide modifications, at least 25,000 unique programmed nucleotide modifications, at least 30,000 unique programmed nucleotide modifications, at least 40,000 unique programmed nucleotide modifications, or at least 50,000 unique programmed nucleotide modifications.

6. The method of claim 1, wherein the plurality of programmed nucleotide modifications that are introduced to the locus of the desired genomic DNA sequence results in a saturating set of programmed nucleotide modifications.

7. The method of claim 1, wherein the plurality of oligonucleotides are synthesized on a microarray or in column-based synthesis.

8. The method of claim 1, wherein the expression system comprises a plasmid which comprises (i) a Cas9 expression cassette that includes a nucleotide sequence which encodes a Cas9 nuclease, (ii) an sgRNA expression cassette, and (iii) a species-specific promoter that is specific to the population of cells.

9. The method of claim 1, further comprising cloning the HDR library prior to the step of co-transfecting the population of cells.

10. The method of claim 1, wherein each oligonucleotide of the HDR library further comprises a pair of homology arms.

11. The method of claim 1, further comprising

(c) harvesting the population of cells,

(d) selectively amplifying a genomic DNA and RNA sample, wherein the edited sequences are amplified and the non-edited sequence are not amplified, and

(e) sequencing the genomic DNA and RNA sample that has been selectively amplified, resulting in a set of genomic transcripts which include the plurality of programmed nucleotide modifications.

12. The method of claim 11, further comprising functionally analyzing the set of genomic transcripts using a functional assay.

13. The method of claim 12, wherein the functional assay is selected from the group consisting of targeted RNA sequencing to measure transcript abundance, targeted DNA sequencing to measure reduced cellular fitness, targeted chromatin immunoprecipitation-sequencing (CHiP-seq) of co-activators to assay enhancers, increased cellular growth rate to assay cancer drivers or drug resistance, and FACS-based phenotypic sorting for cellular assays.

19. A method for genomic screening, comprising:

(a) introducing a plurality of programmed nucleotide modifications to a single genomic locus in a single experiment, wherein step (a) comprises the method of claim 1;

(b) sequencing the genomic DNA or cDNA of the edited locus; and

(c) quantifying the transcript abundance of each mutation.

20. The method of claim 19, wherein step (c) comprises calculating an enrichment score for each mutation.

14. A method for analyzing the functional consequence of a genomic mutation comprising:

(a) synthesizing a homology-directed repair (HDR) library comprising a plurality of oligonucleotides, wherein each oligonucleotide comprises a programmed nucleotide modification in the locus of the desired genome;

(b) co-transfecting a population of cells with (i) an expression system capable of expressing Cas9 and a guide RNA (sgRNA) and (ii) the HDR library, wherein the expression system is capable of introducing the plurality of oligonucleotides having the programmed nucleotide modifications to the locus of the desired genomic DNA sequence in one or more cells of the population;

(c) harvesting the population of cells;

(d) selectively amplifying a genomic DNA and RNA sample, wherein the edited sequences are amplified and the non-edited sequence are not amplified;

(e) sequencing the genomic DNA and RNA sample that has been selectively amplified, resulting in a set of genomic transcripts which include the plurality of programmed nucleotide modifications; and

(f) functionally analyzing the set of genomic transcripts using a functional assay;

wherein the method is carried out in a single experiment.

15. The method of claim 14, wherein the HDR library is constructed using an oligonucleotide comprising a degenerate sequence, wherein the degenerate sequence is between 1 and 100 nucleotides in length.

16. The method of claim 14, wherein the plurality of oligonucleotides are synthesized on a microarray or in column-based synthesis.

17. The method of claim 14, wherein the expression system comprises a plasmid which comprises (i) a Cas9 expression cassette that includes a nucleotide sequence which encodes a Cas9 nuclease, (ii) an sgRNA expression cassette, and (iii) a species-specific promoter that is specific to the population of cells.

18. The method of claim 14, wherein the functional assay is selected from the group consisting of targeted RNA sequencing to measure transcript abundance, targeted DNA sequencing to measure reduced cellular fitness, targeted chromatin immunoprecipitation-sequencing (CHiP-seq) of co-activators to assay enhancers, increased cellular growth rate to assay cancer drivers or drug resistance, and FACS-based phenotypic sorting for cellular assays.