De novo synthesized nucleic acid libraries

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.

What is claimed is: 
     
         1 . A cell library, wherein the cell library comprises a plurality of cell populations, wherein each of the cell populations comprises a DNA molecule encoding for a different gRNA sequence, wherein each gRNA sequence comprises a targeting region for binding to a gene, and wherein at least 15% of the cell populations have at least 2-fold depletion in expression of the gene. 
     
     
         2 . The cell library of  claim 1 , wherein at least 45% of the cell populations have at least 2-fold depletion in expression of the gene. 
     
     
         3 . The cell library of  claim 1 , wherein the gRNA is a single gRNA or a dual gRNA. 
     
     
         4 . The cell library of  claim 1 , wherein the plurality of cell populations comprises DNA molecules encoding for at least 3 different gRNA sequences per a single gene. 
     
     
         5 . The cell library of  claim 1 , wherein the plurality of cell populations comprises DNA molecules encoding for at least 5 different gRNA sequences per a single gene. 
     
     
         6 . The cell library of  claim 1 , wherein the plurality of cell populations comprises at least 2000 cell populations. 
     
     
         7 . The cell library of  claim 1 , wherein the plurality of cell populations comprises DNA molecules encoding for gRNA sequences in a biological pathway. 
     
     
         8 . The cell library of  claim 1 , wherein the plurality of cell populations comprises DNA molecules encoding for gRNA sequences in an entire genome. 
     
     
         9 . The cell library of  claim 8 , wherein the genome is  Arabidopsis thaliana, Caenorhabditis elegans, Canis lupus familiaris, Chlamydomonas reinhardtii, Danio rerio, Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli, Homo sapiens, Macaca mulatta, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus, Saccharomyces cerevisiae , or  Sus scrofa.    
     
     
         10 . The cell library of  claim 1 , wherein each of the cell populations comprises prokaryotic cells. 
     
     
         11 . The cell library of  claim 1 , wherein each of the cell populations comprises eukaryotic cells. 
     
     
         12 . The cell library of  claim 11 , wherein each of the cell populations comprises mammalian cells. 
     
     
         13 . The cell library of  claim 1 , wherein each of the cell populations further comprises an exogenous nuclease enzyme. 
     
     
         14 . The cell library of any one of  claim 1 , wherein the DNA molecule further comprises a vector sequence. 
     
     
         15 . A cell library, wherein the cell library comprises a plurality of cell populations, wherein each of the cell populations comprises a DNA molecule encoding for a different gRNA sequence, wherein each gRNA sequence comprises a targeting region for binding to a gene, and wherein at most 20% of the cell populations have a zero or negative depletion in expression of the gene. 
     
     
         16 . The cell library of  claim 15 , wherein the gRNA is a single gRNA or a dual gRNA. 
     
     
         17 . The cell library of  claim 15 , wherein the plurality of cell populations comprises DNA molecules encoding for at least 3 different gRNA sequences per a single gene. 
     
     
         18 . The cell library of  claim 15 , wherein the plurality of cell populations comprises DNA molecules encoding for at least 5 different gRNA sequences per a single gene. 
     
     
         19 . The cell library of  claim 18 , wherein the plurality of cell populations comprises at least 2000 cell populations. 
     
     
         20 . The cell library of any one of  claim 15 , wherein the plurality of cell populations comprises at least 10000 cell populations. 
     
     
         21 . 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 gRNA;   (b) synthesizing the at least 500 non-identical DNA molecules; and   (c) transcribing the at least 500 non-identical DNA molecules to generate a library of gRNAs, wherein at least about 75% of the gRNAs in the library of gRNAs are error free compared to the predetermined sequences for the at least 500 non-identical DNA molecules.   
     
     
         22 . The method of  claim 21 , further comprising transferring the at least 500 non-identical DNA molecules into cells prior to the transcribing step. 
     
     
         23 . The method of  claim 21 , wherein at least 96% of the gRNAs encoded by the at least 500 non-identical DNA molecules are present in the library of gRNAs. 
     
     
         24 . The method of  claim 21 , wherein at least 87% of the gRNAs in the library of gRNAs are error free compared to the predetermined sequences for the at least 500 non-identical DNA molecules. 
     
     
         25 . The method of  claim 21 , further comprising inserting the at least 500 non-identical DNA molecules into vectors. 
     
     
         26 . The method of  claim 21 , further comprising transferring the at least 500 non-identical DNA molecules to cells of an organism. 
     
     
         27 . The method of  claim 26 , wherein the organism is  Arabidopsis thaliana, Caenorhabditis elegans, Canis lupus familiaris, Chlamydomonas reinhardtii, Danio rerio, Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli, Homo sapiens, Macaca mulatta, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus, Saccharomyces cerevisiae , or  Sus scrofa.    
     
     
         28 . The method of  claim 21 , wherein each non-identical DNA molecule encodes for a single gRNA or a dual gRNA.