De novo synthesized nucleic acid libraries

• US 10,053,688 B2
• Filed: 08/21/2017
• Issued: 08/21/2018
• Est. Priority Date: 08/22/2016
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Cox, Anthony, Chen, Siyuan
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/682,100
• Publication Number:
  US 20180051278A1
• Time in Patent Office: 365 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

Disclosed herein are methods for the generation of nucleic acid libraries encoding for gRNA sequences. The gRNAs encoded by methods described herein may be single or double gRNA sequences. Methods described provide for the generation of gRNA libraries, as a DNA precursor or as a RNA transcription product, with improved accuracy and uniformity.

Claims

1. A nucleic acid library, wherein the nucleic acid library comprises at least 500 non-identical DNA molecules, wherein each non-identical DNA molecule encodes for a different gRNA sequence, wherein each gRNA sequence comprises a targeting domain complementary to a mammalian gene, and wherein at least about 80% of the at least 500 non-identical DNA molecules are each present in the nucleic acid library in an amount within 2×

of a mean frequency for each of the non-identical DNA molecules in the library.

2. The nucleic acid library of claim 1, wherein each non-identical DNA molecule has a GC base content of about 20% to about 85%.

3. The nucleic acid library of claim 1, wherein each non-identical DNA molecule has a GC base content of about 30% to about 70%.

4. The nucleic acid library of claim 1, wherein at least about 90% of the at least 500 non-identical DNA molecules are each present in the nucleic acid library in an amount within 2×

of the mean frequency for each of the non-identical DNA molecules in the library.

5. The nucleic acid library of claim 1, wherein at least 99% of the at least 500 non-identical DNA molecules are each present in the nucleic acid library in an amount within 2×

of the mean frequency for each of the non-identical DNA molecules in the library.

6. The nucleic acid library of claim 1, wherein the at least 500 non-identical DNA molecules comprise at least 2000 non-identical DNA molecules.

7. The nucleic acid library of claim 1, wherein the at least 500 non-identical DNA molecules comprise at least 3500 non-identical DNA molecules.

8. The nucleic acid library of claim 1, wherein the at least 500 non-identical DNA molecules comprise at least 100,000 non-identical DNA molecules.

9. The nucleic acid library of claim 1, wherein each non-identical DNA molecule comprises up to 200 bases in length.

10. The nucleic acid library of claim 1, wherein each non-identical DNA molecule comprises about 100 to about 200 bases in length.

11. The nucleic acid library of claim 1, wherein the at least 500 non-identical DNA molecules comprise non-identical DNA molecules encoding for gRNA sequences targeting genes in a biological pathway.

12. The nucleic acid library of claim 1, wherein the at least 500 non-identical DNA molecules comprise non-identical DNA molecules encoding for gRNA sequences targeting genes in an entire genome.

13. The nucleic acid library of claim 1, wherein the gRNA is a single gRNA or a dual gRNA.

14. A nucleic acid library, wherein the nucleic acid library comprises at least 2000 non-identical nucleic acids, wherein each non-identical nucleic acid encodes for a different gRNA sequence, wherein each gRNA sequence comprises a targeting domain complementary to a eukaryotic gene, and wherein at least about 80% of the at least 2000 non-identical nucleic acids are present in the nucleic acid library in an amount within 2×

of a mean frequency for each of the non-identical nucleic acids in the library.

15. The nucleic acid library of claim 14, wherein each non-identical nucleic acid has a GC base content of about 20% to about 85%.

16. The nucleic acid library of claim 14, wherein each non-identical nucleic acid has a GC base content of about 30% to about 70%.

17. The nucleic acid library of claim 14, wherein at least about 90% of the at least 2000 non-identical nucleic acids are each present in the nucleic acid library in an amount within 2×

of the mean frequency for each of the non-identical nucleic acids in the library.

18. The nucleic acid library of claim 14, wherein at least 99% of the at least 2000 non-identical nucleic acids are each present in the nucleic acid library in an amount within 2×

of the mean frequency for each of the non-identical nucleic acids in the library.

19. The nucleic acid library of claim 14, wherein each non-identical nucleic acid comprises up to 200 bases in length.

20. The nucleic acid library of claim 14, wherein each non-identical nucleic acid comprises about 100 to about 200 bases in length.

21. The nucleic acid library of claim 14, wherein the at least 2000 non-identical nucleic acids comprise non-identical nucleic acids encoding for gRNA sequences targeting genes in a biological pathway.

22. The nucleic acid library of claim 14, wherein the at least 2000 non-identical nucleic acids comprise non-identical nucleic acids encoding for gRNA sequences targeting genes in an entire genome.

23. The nucleic acid library of claim 14, wherein each non-identical nucleic acid comprises DNA or RNA molecules.

24. An amplicon library, wherein the amplicon library comprises a plurality of non-identical DNA molecules, wherein each non-identical DNA molecule is present in a population of amplification products, wherein each non-identical DNA molecule encodes for a different gRNA sequence, wherein each gRNA sequence comprises a targeting domain complementary to a eukaryotic gene, and wherein at least about 80% of the plurality of non-identical DNA molecules are each present in the amplicon library in an amount within 2×

of a mean frequency for each of the non-identical DNA molecules in the library.

25. The amplicon library of claim 24, wherein each non-identical DNA molecule has a GC base content of about 30% to about 70%.

26. The amplicon library of claim 24, wherein the gRNA is a single gRNA or a dual gRNA.