De novo synthesized nucleic acid libraries

Patent metadata

Field	Value
Publication number	US-10975372-B2
Application number	US-201816031784-A
Country	US
Kind code	B2
Filing date	Jul 10, 2018
Priority date	Aug 22, 2016
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

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.

First claim

What is claimed is:

1. A method for synthesis of a gRNA library, comprising:
(a) providing predetermined sequences for at least 500 non-identical DNA molecules, wherein each non-identical DNA molecule encodes for a different gRNA;
(b) providing a surface, wherein the surface comprises clusters of loci for nucleic acid extension reaction;
(c) synthesizing a nucleic acid library comprising the at least 500 non-identical DNA molecules, wherein each non-identical DNA molecule extends from the surface, 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; and
(d) transferring the at least 500 non-identical DNA molecules into cells capable of generating the gRNA library transcribed from the at least 500 non-identical DNA molecules.

2. The method of claim 1 , wherein each of the clusters comprises about 50 to about 500 loci.

3. The method of claim 1 , wherein each non-identical DNA molecule comprises up to about 200 bases in length.

4. The method of claim 1 , wherein each non-identical DNA molecule encodes for a single gRNA or a dual gRNA.

5. The method of claim 1 , wherein the cells are prokaryotic cells.

6. The method of claim 1 , wherein the cells are eukaryotic cells.

7. The method of claim 1 , wherein the cells are mammalian cells.

8. The method of claim 1 , wherein each of the cells comprises an exogenous nuclease enzyme.

9. The method of claim 1 , wherein each non-identical DNA molecule further comprises a vector sequence.

10. The method of claim 1 , wherein each non-identical DNA molecule has a GC base content of 20% to 85%.

11. The method of claim 1 , wherein each non-identical DNA molecule has a GC base content of 30% to 70%.

12. The method of claim 1 , wherein at least 96% of the gRNAs encoded by the at least 500 non-identical DNA molecules are present in the gRNA library.

13. The method of claim 1 , wherein the at least 500 non-identical DNA molecules comprises at least 2000 non-identical DNA molecules.

14. The method of claim 1 , wherein the at least 500 non-identical DNA molecules comprises at least 100,000 non-identical DNA molecules.

15. The method of claim 1 , wherein the gRNA targets genes in a biological pathway.

16. The method of claim 1 , wherein the gRNA targets genes in an entire genome.

Assignees

• Twist Bioscience Corp

Inventors

• Cox Anthony
• Chen Siyuan

Classifications

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N2330/31

  Libraries, arrays · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title