WO2018038772A1 - De novo synthesized nucleic acid libraries - Google Patents

De novo synthesized nucleic acid libraries Download PDF

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Publication number
WO2018038772A1
WO2018038772A1 PCT/US2017/026232 US2017026232W WO2018038772A1 WO 2018038772 A1 WO2018038772 A1 WO 2018038772A1 US 2017026232 W US2017026232 W US 2017026232W WO 2018038772 A1 WO2018038772 A1 WO 2018038772A1
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library
nucleic acid
grna
dna molecules
instances
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French (fr)
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Anthony Cox
Siyuan CHEN
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Twist Bioscience Corp
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Twist Bioscience Corp
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Priority to AU2017315294A priority Critical patent/AU2017315294B2/en
Priority to GB1903914.8A priority patent/GB2568444A/en
Priority to KR1020197008458A priority patent/KR102212257B1/ko
Priority to CN201780065373.9A priority patent/CN109996876A/zh
Priority to EA201990487A priority patent/EA201990487A1/ru
Priority to EP17844060.8A priority patent/EP3500672A4/en
Priority to SG11201901563UA priority patent/SG11201901563UA/en
Priority to JP2019510673A priority patent/JP6854340B2/ja
Application filed by Twist Bioscience Corp filed Critical Twist Bioscience Corp
Priority to CA3034769A priority patent/CA3034769A1/en
Publication of WO2018038772A1 publication Critical patent/WO2018038772A1/en
Priority to IL264966A priority patent/IL264966B/en
Anticipated expiration legal-status Critical
Priority to AU2024200331A priority patent/AU2024200331A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B40/00—Processes, in general, for influencing or modifying the properties of mortars, concrete or artificial stone compositions, e.g. their setting or hardening ability
    • C04B40/06—Inhibiting the setting, e.g. mortars of the deferred action type containing water in breakable containers ; Inhibiting the action of active ingredients
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays
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    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—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

Definitions

  • the cornerstone of synthetic biology is the design, build, and test process— an iterative process that requires DNA to be made accessible for rapid and affordable generation and optimization of these custom pathways and organisms.
  • the A, C, T, and G nucleotides that constitute DNA are formulated into the various sequences that would comprise a region of interest, with each sequence variant representing a specific hypothesis that will be tested.
  • These variant sequences represent subsets of sequence space, a concept that originated in evolutionary biology and pertains to the totality of sequences that make up genes, genomes, transcriptome, and proteome.
  • targeted genome editing there exists a need for rapid generation of highly accurate and uniform nucleic acid libraries for specifically directing enzymatic editing of a gene, a gene cluster, a pathway, or an entire genome.
  • Nucleic acid libraries as described herein comprise nucleic acids for specifically targeting and editing a gene, a gene cluster, a biological pathway, or an entire genome.
  • nucleic acid libraries 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, 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 2x of a mean frequency for each of the non-identical DNA molecules in the library.
  • nucleic acid libraries wherein each non-identical DNA molecule has a GC base content of about 20% to about 85%).
  • nucleic acid libraries wherein each non-identical DNA molecule has a GC base content of about 30%> to about 70%.
  • nucleic acid libraries 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 2x of the mean frequency for each of the non-identical DNA molecules in the library.
  • nucleic acid libraries 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 2x of the mean frequency for each of the non-identical DNA molecules in the library.
  • nucleic acid libraries wherein the at least 500 non-identical DNA molecules comprises at least 2000 non-identical DNA molecules.
  • nucleic acid libraries wherein the at least 500 non-identical DNA molecules comprises at least 3500 non-identical DNA molecules.
  • nucleic acid libraries wherein the at least 500 non-identical DNA molecules comprises at least 100,000 non-identical DNA molecules.
  • nucleic acid libraries wherein each non-identical DNA molecule comprises up to 200 bases in length.
  • nucleic acid libraries wherein the at least 500 non-identical DNA molecules comprises non-identical DNA molecules encoding for gRNA sequences targeting genes in a biological pathway.
  • nucleic acid libraries wherein the at least 500 non-identical DNA molecules comprises non-identical DNA molecules encoding for gRNA sequences targeting genes in an entire genome.
  • nucleic acid libraries wherein the gRNA is a single gRNA or a dual gRNA.
  • nucleic acid libraries wherein the nucleic acid library comprises at least 2000 non-identical nucleic acids, wherein each non-identical nucleic acid encodes for a different sgRNA sequence, wherein each sgRNA 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 2x of a mean frequency for each of the non-identical nucleic acids in the library.
  • nucleic acid libraries wherein each non-identical nucleic acid has a GC base content of about 20% to about 85%).
  • nucleic acid libraries wherein each non-identical nucleic acid has a GC base content of about 30%> to about 70%.
  • nucleic acid libraries 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 2x of the mean frequency for each of the non-identical nucleic acids in the library.
  • nucleic acid libraries 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 2x of the mean frequency for each of the non-identical nucleic acids in the library.
  • nucleic acid libraries wherein each non-identical nucleic acid comprises up to 200 bases in length.
  • nucleic acid libraries wherein each non-identical nucleic acid comprises about 100 to about 200 bases in length.
  • nucleic acid libraries wherein the at least 2000 non-identical nucleic acids comprise non-identical nucleic acids encoding for sgRNA sequences targeting genes in a biological pathway.
  • nucleic acid libraries wherein the at least 2000 non-identical nucleic acids comprise non-identical nucleic acids encoding for sgRNA sequences targeting genes in an entire genome.
  • each non-identical nucleic acid comprises DNA or RNA molecules.
  • amplicon libraries wherein the amplicon library comprises a plurality of non-identical DNA molecules, wherein each non-identical DNA is present in a population of amplification products, wherein each non-identical DNA molecule encodes for a different gRNA sequence, 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 2x of a mean frequency for each of the non-identical DNA molecules in the library.
  • amplicon libraries wherein each non-identical DNA molecule has a GC base content of about 30% to about 70%.
  • amplicon libraries wherein the gRNA is a single gRNA or a dual gRNA.
  • cell libraries 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.
  • cell libraries wherein at least 45% of the cell populations have at least 2-fold depletion in expression of the gene.
  • the gRNA is a single gRNA or a dual gRNA.
  • cell libraries wherein the plurality of cell populations comprises DNA molecules encoding for at least 3 different gRNA sequences per a single gene.
  • cell libraries wherein the plurality of cell populations comprises DNA molecules encoding for at least 5 different gRNA sequences per a single gene.
  • cell libraries wherein the plurality of cell populations comprises at least 2000 cell populations.
  • cell libraries wherein the plurality of cell populations comprises DNA molecules encoding for gRNA sequences in a biological pathway.
  • the plurality of cell populations comprises DNA molecules encoding for gRNA sequences in an entire genome.
  • cell libraries 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.
  • each of the cell populations comprises prokaryotic cells.
  • each of the cell populations comprises eukaryotic cells.
  • each of the cell populations comprises mammalian cells.
  • each of the cell populations further comprises an exogenous nuclease enzyme.
  • the DNA molecule further comprises a vector sequence.
  • cell libraries 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.
  • the gRNA is a single gRNA or a dual gRNA.
  • the plurality of cell populations comprises DNA molecules encoding for at least 3 different gRNA sequences per a single gene.
  • the plurality of cell populations comprises DNA molecules encoding for at least 5 different gRNA sequences per a single gene.
  • the plurality of cell populations comprises at least 2000 cell populations.
  • cell libraries wherein the plurality of cell populations comprises at least 10000 cell populations.
  • gRNA library comprising: providing predetermined sequences for at least 500 non-identical DNA molecules, wherein each non-identical DNA molecule encodes for a gRNA; synthesizing the at least 500 non-identical DNA molecules; and 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.
  • methods for synthesis of a gRNA library further comprising transferring the at least 500 non- identical DNA molecules into cells prior to the transcribing step.
  • gRNA library wherein the organism is Arabidopsis thaliana, Caenorhabditis elegans, Canis lupus familiar is, 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.
  • methods for synthesis of a gRNA library wherein each non-identical DNA molecule encodes for a single gRNA or a dual gRNA.
  • gRNA library comprising: providing predetermined sequences for a plurality of non-identical DNA molecules, wherein each non- identical DNA molecule encodes for a gRNA; providing a surface, wherein the surface comprises clusters of loci for nucleic acid extension reaction; synthesizing the plurality of non-identical DNA molecules, wherein each non-identical DNA molecule extends from the surface; and transferring the plurality of non-identical DNA molecules into cells.
  • each cluster comprises about 50 to about 500 loci.
  • each non-identical DNA molecule comprises up to about 200 bases in length.
  • methods for synthesis of a gRNA library wherein each non-identical DNA molecule encodes for a single gRNA or a dual gRNA.
  • methods for synthesis of a gRNA library wherein the cells are prokaryotic cells.
  • methods for synthesis of a gRNA library wherein the eukaryotic are mammalian cells.
  • methods for synthesis of a gRNA library, wherein each of the cells comprises an exogenous nuclease enzyme.
  • FIG. 1A illustrates a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complex which includes the following components: PAM, target sequence, CAS9 enzyme, Guide RNA (gRNA), and donor DNA.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • FIG. 1B illustrates a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complex which includes the following components: PAM, target sequence, CAS9 enzyme, Guide RNA (gRNA), and donor DNA for a non-homologous end joining repair (NHEJ) pathway.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Figure 2 illustrates a gRNA library screening workflow, including design, synthesis, cloning, packaging, screening and analysis of a gRNA library.
  • Figure 3 illustrates gRNA library screening workflow for building a library, including: synthesizing an oligonucleic acid library on an array, amplifying and transferring the oligonucleic acids into vectors, and forming an expression library for gRNA expression.
  • Figures 4A-4C are diagrams of various gRNAs.
  • Figure 4A is diagram of a sgRNA sequence (SEQ ID NO: 40) having a base-pairing region, a dCas9 handle, and a S. pyogenes terminator region.
  • Figure 4B is a diagram of a sgRNA alone.
  • Figure 4C is a diagram of a dgRNA alone.
  • Figure 5A is a diagram of a sgRNA sequence in a template strand targeting
  • Figure 5B is a diagram of a sgRNA sequence in a non-template strand targeting arrangement.
  • Figure 6A is a diagram of a gRNA sequence with a T7 promoter that, when transcribed, results in gRNA sequence that forms hairpin secondary structure.
  • Figure 6B is a diagram of a gRNA sequence with a T7 promoter that, when transcribed, results in gRNA sequence that does not form a hairpin secondary structure.
  • Figure 7 depicts a workflow for in vitro Cas9 mediated cleavage of target DNA.
  • Figure 8 illustrates an example of a computer system.
  • Figure 9 is a block diagram illustrating an example architecture of a computer system.
  • Figure 10 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).
  • NAS Network Attached Storage
  • Figure 11 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.
  • Figure 12 depicts 4 sgRNA designs.
  • Figure 12 discloses SEQ ID NOS 20, 15, 21, 15,
  • Figures 13A-13B are plots from a BioAnalyzer reading, with nucleotide bases on the X axis and fluorescent units on the Y axis.
  • Figures 14A-14J are plots from a BioAnalyzer reading, with nucleotide bases on the X axis and fluorescent units on the Y axis.
  • Figure 15 is an image of a 256 clusters, each cluster having 121 loci with oligonucleic acids extending therefrom.
  • Figure 16A is a plot of oligonucleic acid representation (oligonucleic acid frequency v. absorbance) across a plate from synthesis of 29,040 unique oligonucleic acids from 240 clusters, each cluster having 121 oligonucleic acids.
  • Figure 16B is a plot of measurement of oligonucleic acid frequency v. absorbance across each individual cluster, with control clusters identified by a box.
  • Figure 17 is a plot of measurements of oligonucleic acid frequency v. absorbance across four individual clusters.
  • Figure 18A is a plot of on error rate v. frequency across a plate from synthesis of 29,040 unique oligonucleic acids from 240 clusters, each cluster having 121 oligonucleic acids.
  • Figure 18B is a plot of measurement of oligonucleic acid error rate v. frequency across each individual cluster, with control clusters identified by a box.
  • Figure 19 is a plot of measurements of oligonucleic acid error rate v. frequency across four clusters.
  • Figure 20 is a plot of GC content as a measure of percent per oligonucleic acid v. the number of oligonucleic acids.
  • Figure 21 provides plots with results from PCR with two different polymerases. Each chart depicts "observed frequency" ("0 to 35" measured in counts per 100,000) v. number of oligonucleic acids (0 to 2000).
  • Figure 22 provides a chart with quantification of oligonucleic acid population uniformity post amplification that was recorded.
  • Figure 23 depicts a plot of impact of over amplification on sequence dropouts.
  • Figures 24A-24B depict results from sequencing recovered oligonucleic acids from a 10,000 sgRNA oligonucleic acid CRISPR library.
  • Figure 25 depicts results from sequencing recovered oligonucleic acids from a 101,000 sgRNA oligonucleic acid CRISPR library.
  • Figure 26A depicts a graph of percentage of sgRNAs with at least 2-fold depletion.
  • Figure 26B depicts a graph of percentage of sgRNAs with zero or negative depletion.
  • gRNA guide RNA
  • gRNA refers to guide RNA sequence and encompasses both single and dual guide RNA sequence.
  • dgRNA refers to dual guide RNA sequence: crRNA (spacer sequence comprising a seed region complementary to a target sequence) and a separate tracrRNA (trans-activating sequence), which are partially complementary RNAs.
  • sgRNA refers to single guide RNA sequence, comprising both a fused crRNA and tracrRNA.
  • oligonucleic acid and nucleic acid encompass double- or triple-stranded nucleic acids, as well as single-stranded molecules.
  • the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).
  • Nucleic acid sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids.
  • oligonucleic acid and “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length.
  • amplicon refers to an amplification reaction product.
  • gRNA libraries generated using methods described herein include both sgRNA and dgRNA libraries.
  • methods for highly uniform synthesis resulting in high representation of predetermined gRNAs in the resulting libraries are designed. See FIG. 2.
  • Design strategies include, without limitation, design of gRNAs to span a gene.
  • the de novo synthesized nucleic acids are DNA or RNA bases.
  • a library comprising nucleic acids is synthesized, wherein each nucleic acid synthesized is a DNA sequence that encodes for a gRNA (e.g., sgRNA) sequence as a transcription product.
  • the synthesized nucleic acids are then inserted into expression vectors.
  • the synthesized nucleic acids are inserted into viral vectors, and then packaged for transduction into cells, followed by screening and analysis.
  • Exemplary cells include without limitation, prokaryotic and eukaryotic cells.
  • Exemplary eukaryotic cells include, without limitation, animal, plant, and fungal cells.
  • Exemplary animal cells include, without limitation, insect, fish and mammalian cells.
  • Exemplary mammalian cells include mouse, human, and primate cells.
  • Exemplary cellular functions tested include, without limitation, changes in cellular proliferation, migration/adhesion, metabolic, and cell-signaling activity.
  • the gRNA itself is synthesized and available for downstream applications, such as transfection into cells.
  • Oligonucleic acids may be synthesized within a cluster 303 of locations ("loci") for extension on an array 301. See FIG. 3. Such an arrangement may provide for improved oligonucleic acid representation of products from amplification of the synthesized oligonucleic acids -termed "amplicons"-when compared to amplification products of oligonucleic acids synthesized across an entire plate without a clustered loci arrangement.
  • amplification 310 of oligonucleic acids synthesized within a single cluster counters negative effects on representation due to repeated synthesis of large oligonucleic acid populations having oligonucleic acids with heavy GC content, commonly termed "drift," due to underrepresentation of GC low or GC high amplicons in the amplification reaction product.
  • the single cluster described herein comprises about 50-1000, 75-900, 100-800, 125-700, 150-600, 200-500, or 300-400 discrete loci.
  • the single cluster comprises 50-500 discrete loci.
  • a locus is a spot, well, microwell, channel, or post.
  • each cluster has at least IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, or more redundancy of separate features supporting extension of oligonucleic acids having identical sequence.
  • an array 301 includes multiple clusters 303 of loci for oligonucleic acid synthesis and extension.
  • De novo DNA is synthesized and removed from the plate to form a population of oligonucleic acids 305 (e.g., DNAs encoding for sgRNAs), which are subject to amplification 310 to form a library of amplified oligonucleic acids 320 for insertion into a vector 330 to form a library of vectors including the synthesized DNAs 335.
  • the DNAs are transcribed into gRNAs (e.g., sgRNAs) and are available for binding with genomic editing regime (e.g., a Cas9-based system).
  • the cells may have natural or ectopic expression of the editing enzyme (e.g., Cas9).
  • the editing enzyme e.g., Cas9
  • the editing enzyme may have double DNA strand cleavage activity, or a modified activity, such as nicking, base swapping or sequence swapping activity.
  • the synthesized DNA for insertion into a vector may comprise sgRNAs, dgRNAs, or fragments thereof.
  • Expression vectors for inserting nucleic acid libraries disclosed herein comprise eukaryotic or prokaryotic expression vectors.
  • Exemplary expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO- COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl Vector, pEFla-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors:
  • De novo oligonucleic acid libraries synthesized by methods described herein may be expressed in cells.
  • the cells are associated with a disease state.
  • cells associated with a disease state include, but not limited to, cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system.
  • the model system is a plant or animal system.
  • the de novo oligonucleic acid libraries synthesized by methods described herein may be expressed in cells.
  • the cells are associated with a disease state.
  • cells associated with a disease state include, but not limited to, cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system.
  • the model system is a plant or animal system.
  • the de novo is a plant or animal system.
  • oligonucleic acid libraries are expressed in cells to assess for a change in cellular activity.
  • Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity, aging, response to free radical damage, or any combination thereof.
  • gRNA library or a DNA library that when transcribed results in a gRNA library
  • the gRNA library comprises a plurality of non-identical gRNAs per a gene.
  • the gRNA may encode a sgRNA or a dgRNA.
  • the gRNA library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical gRNAs per the gene.
  • the gRNA library targets one or more genes. In some instances, the gRNA library targets about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes.
  • the gRNA library targets about 1-100, 2-95, 5-90, 10-85, 15-80, 20- 75, 25-70, 30-65, 35-60, or 40-50 genes.
  • the gRNA library described herein targets genes in a pathway. Exemplary pathways include, without limitation a metabolic, cell death, cell cycle progression, immune cell activation, inflammatory response, angiogenesis, lymphogenesis, hypoxia and oxidative stress response, cell adhesion, and cell migration pathways.
  • Methods for synthesizing a gRNA library as described herein may provide for synthesis of non-identical gRNAs having a base-pairing region complementary to part of a genome, a genome target region.
  • the genome target region may comprise exon, intron, coding, or non-coding sequence.
  • the gRNA library comprises non-identical gRNAs collectively having a base-pairing region complementary to at least or about 5% of the genes in an entire genome.
  • the gRNA library comprises non-identical gRNAs collectively having a base- pairing region complementary to at least or about 80% of the genes in an entire genome.
  • the gRNA library comprises non-identical gRNAs collectively having a base-pairing region complementary to at least or about 90% of the genes in an entire genome. In some instances, the gRNA library comprises non-identical gRNAs collectively having a base-pairing region complementary to at least or about 95% of the genes in an entire genome. In some instances, the gRNA library comprises non-identical gRNAs collectively having a base-pairing region complementary to at least or about 100% of the genes in an entire genome.
  • gRNA libraries synthesized by methods described herein that result in gRNAs with at least 2X depletion of a gene across different cells.
  • the gRNA libraries comprise at least or about 10%, 12%, 15%, 16%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%), 60%), 65% or more of gRNAs that provide for at least 2X depletion a gene when present in cells or in a plurality of cell populations.
  • the gene is an essential gene, i.e. a gene critical for cell survival.
  • Exemplary essential genes include, without limitation, PCNA, PSMA7, RPP21, and SF3B3.
  • the gRNA libraries comprise gRNAs that provide for at least 2X, 3X, 4X, 5X, 6X, or more than 6X depletion of a gene when present in cells.
  • the gRNA libraries comprise at most 5%>, 10%>, 12%>, 15%>, or 20%> of the gRNAs with zero or negative depletion of the gene when present in cells or in a plurality of cell populations.
  • the plurality of cell populations comprises at least or about 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 15000, 20000, 25000, 30000, or more than 30000 cell populations.
  • the gRNA libraries comprise gRNAs with at least 2X, 3X, 4X, 5X, 6X, or more than 6X depletion for the plurality of genes. In some instances, the gRNA libraries comprise an average of at least or about 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%) or more than 90%> of gRNAs providing at least 2X depletion for the plurality of genes.
  • the gRNAs providing such gene deletion profiles can be sgRNAs or dgRNAs.
  • RNA or DNA synthesized oligonucleic acids
  • 4X of the mean for oligonucleic acid representation for a nucleic acid library.
  • more than 90%> of oligonucleic acids are represented within 2X of the mean for oligonucleic acid representation for the library.
  • more than 90%> of oligonucleic acids are represented within 1.5X of the mean for oligonucleic acid representation for the library.
  • more than 80%> of oligonucleic acids are represented within 1.5X of the mean for oligonucleic acid representation for the library.
  • Oligonucleic acid libraries de novo synthesized by methods described herein comprise a high percentage of correct sequences compared to predetermined sequences.
  • de novo oligonucleic acids libraries disclosed herein have greater than 70%> correct sequence compared to predetermined sequences for oligonucleic acids.
  • de novo oligonucleic acids libraries disclosed herein have greater than 75%> correct sequence compared to predetermined sequences for the oligonucleic acids.
  • de novo oligonucleic acids libraries disclosed herein have greater than 80% correct sequence compared to predetermined sequences for the oligonucleic acids.
  • de novo oligonucleic acids libraries disclosed herein have greater than 85%> correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo oligonucleic acids libraries disclosed herein have greater than 90% correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo oligonucleic acids libraries disclosed herein have greater than 95% correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo oligonucleic acids libraries disclosed herein have greater than 100%> correct sequence compared to predetermined sequences for the oligonucleic acids.
  • de novo synthesized oligonucleic acids libraries disclosed herein have greater than 70% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein have greater than 75% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein have greater than 80% correct sequence compared to predetermined sequences for the oligonucleic acids following an
  • de novo synthesized oligonucleic acids libraries disclosed herein have greater than 85% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein have greater than 90% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein have greater than 95% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein have 100% correct sequence compared to predetermined sequences for the oligonucleic acids following an amplification reaction.
  • de novo synthesized oligonucleic acids libraries disclosed herein when transferred into cells, results in greater than 80% correct sequence compared to
  • de novo synthesized oligonucleic acids libraries disclosed herein when transferred into cells, results in greater than 85% correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, results in greater than 90% correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, results in greater than 95% correct sequence compared to predetermined sequences for the oligonucleic acids. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, results in 100% correct sequence compared to predetermined sequences for the oligonucleic acids.
  • de novo synthesized oligonucleic acids libraries disclosed herein when transferred into cells, result in greater than 80% sequence representation. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, result in greater than 90% sequence representation. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, result in greater than 95% sequence representation. In some instances, de novo synthesized oligonucleic acids libraries disclosed herein, when transferred into cells, result in 100% sequence representation.
  • De novo oligonucleic acid libraries described herein may be subject to amplification reactions with the addition of a polymerase enzyme and amplification reagents (e.g., buffers, phosphates, and dNTPs).
  • a polymerase enzyme and amplification reagents e.g., buffers, phosphates, and dNTPs.
  • the de novo oligonucleic acid libraries are amplified by PCR for at least or about 6, 8, 10, 15, 20, or more than 20 cycles.
  • the de novo oligonucleic acid libraries are amplified by PCR in a range of about 6 to 20, 7 to 18, 8 to 17, 9 to 16, or 10 to 15 cycles.
  • the de novo oligonucleic acid libraries are amplified by PCR for about 15 cycles.
  • amplification of the de novo oligonucleic acid libraries provides for an amplicon library of DNA molecules.
  • the amplicon library comprises non- identical nucleic acids that encode for a gRNA sequence.
  • the gRNA sequence is a sgRNA or a dgRNA.
  • the de novo oligonucleic acid libraries comprise non-identical nucleic acids, wherein each non-identical nucleic acid comprises DNA molecules.
  • the number of DNA molecules is about 500, 2000, 3500 or more molecules. In some instances, the number of DNA molecules is at least or about 250, 500, 1000, 1250, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 15000, 20000, 50000, 100000, 250000, 500000, 750000, 1 million, or more than 1 million molecules.
  • the number of DNA molecules is at most 250, 500, 1000, 1250, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 15000, 20000, 50000, 100000, 250000, 500000, 750000, 1 million, or more than 1 million molecules.
  • the DNA molecule encodes for a gRNA sequence.
  • the gRNA sequence is a sgRNA or a dgRNA.
  • the de novo oligonucleic acid libraries comprise non-identical nucleic acids, wherein each non-identical nucleic acid comprises RNA molecules.
  • the number of RNA molecules is about 2000 molecules. In some instances, the number of RNA molecules is at least or about 250, 500, 1000, 1250, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 15000, 20000, 50000, 100000, 250000, 500000, 750000, 1 million, or more than 1 million molecules.
  • the number of RNA molecules is at most 250, 500, 1000, 1250, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 15000, 20000, 50000, 100000, 250000, 500000, 750000, 1 million, or more than 1 million molecules.
  • the RNA molecule encodes for a gRNA sequence.
  • the gRNA sequence is a sgRNA or a dgRNA.
  • de novo oligonucleic acid libraries having high uniformity following amplification.
  • more than 80% of oligonucleic acids in a de novo oligonucleic acid library described herein are represented within at least about 1.5X the mean representation for the entire library following amplification.
  • more than 90% of oligonucleic acids in a de novo oligonucleic acid library described herein are represented within at least about 1.5X the mean representation for the entire library following amplification.
  • more than 80% of oligonucleic acids in a de novo oligonucleic acid library described herein are represented within at least about 2X the mean representation for the entire library following amplification.
  • more than 80% of oligonucleic acids in a de novo oligonucleic acid library described herein are represented within at least about 2X the mean representation for the entire library following amplification.
  • An unamplified population of oligonucleic acids de novo synthesized using methods described herein can vary in a number of non-identical oligonucleic acid sequences.
  • the number of non -identical oligonucleic acid sequences is in a range of about 2000-1 million, 3000 to 900000, 4000-800000, 5000-700000, 6000-600000, 7000-500000, 8000-400000, 9000-300000, 10000-200000, 11000-100000, 12000-75000, 14000-60000, and 20000-50000 sequences.
  • the number of non-identical oligonucleic acid sequences is in the range of about 50-2000, 75-1800, 100-1700, 150-1600, 200-1500, 250-1400, 300-1300, 400-1200, 500- 1100, 600-1000, 700-900 sequences. In some instances, the number of non-identical oligonucleic acid sequences is 2000 sequences. In some instances, the number of non-identical oligonucleic acid sequences is more than 1 million sequences.
  • the number of non-identical oligonucleic acid sequences is at least 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 3000, 5000, 7000, 10000, 20000, 30000, 50000, 100000, 500000, 700000, 1000000, 10000000, 1000000000, or more sequences. In some instances, the number of non-identical oligonucleic acids sequence is up to 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 3000, 5000, 7000, 10000, 20000, 30000, 50000, 100000, 500000, 700000, 1000000, or more sequences.
  • the number of non-identical oligonucleic acid sequences is at most 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 3000, 5000, 7000, 10000, 20000, 30000, 50000, 100000, 500000, 700000, and 1000000 sequences.
  • An oligonucleic acid of an unamplified population may be present in varying amounts. In some instances, an oligonucleic acid of an unamplified population is present in an amount of at least or about 0.25 femtomole. In some instances, an oligonucleic acid of an unamplified population is present in an amount of at least or about 1 femtomole. In some instances, an oligonucleic acid of an unamplified population is present in an amount of at least 0.25, 1, 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, or more than 1000 femtomoles. In some instances, an oligonucleic acid of an unamplified population is present in an amount of at most 0.25, 1, 10, 20, 30, 40, 50, 100, 250, 500, 750, and 1000 femtomoles.
  • sequence length or average sequence length of the non-identical oligonucleic acids vary.
  • the sequence length or average sequence length of the non-identical oligonucleic acids is up to 150 bases.
  • sequence length or average sequence length of the non-identical oligonucleic acids is in a range of about 100 to about 200 bases.
  • the sequence length or average sequence length of the non-identical oligonucleic acids is at least 30, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or more than 500 bases.
  • sequence length or average sequence length of the non-identical oligonucleic acids is at most 150, 200, 250, 300, 350, 400, 450, or 500 bases.
  • An exemplary sequence length of the non-identical oligonucleic acid is in a range of about 25 to about 150 or about 50 to about 200 bases. In some cases, the sequence length or average sequence length of the non-identical oligonucleic acids is in the range of about 125 to about 200 or about 150 to about 200 bases.
  • sgRNA single guide RNA
  • Cas9 genomic sequence editing enzyme
  • FIG. 4A An example sgRNA in complex with a Cas9 enzyme is illustrated in FIG. 4A, and an example alone in FIG. 4B.
  • the gRNA may be a dual guide RNA, as illustrated in FIG. 4C.
  • Guide sequences disclosed herein comprises a base-pairing region.
  • the base-pairing region comprises a seed region for binding to a target sequence and, optionally, a spacer region.
  • the base-pairing region may vary in length.
  • the base-pairing region may comprise about 1 to 10, 1 to 20, 20 to 25, or 1 to 30 bases in length.
  • the base-pairing region comprises at least 10, 15, 20, 25, 30 or more bases in length. In some instances, the base-pairing region comprises a seed region of at least 10 bases in length. The seed region may comprise about 8 to 20 bases in length. In some instances, the seed region is about 12 bases in length. In some instances, a base-pairing region described herein is designed to target a template strand during transcription, FIG. 5A. In some instances, a base-pairing region described herein is designed to target a non-template strand during transcription, FIG. 5B.
  • 3' of the base-pairing region of a sgRNA is a Cas9 handle region for binding to Cas9.
  • the Cas9 handle region is a dCas9 handle region for binding to a dCas9 enzyme.
  • the handle region may vary in length.
  • the handle region may comprise about 1 to 50, 20 to 45, or 15 to 60 bases in length.
  • the handle region comprises at least 35, 40, 45, 50 or more bases in length.
  • the handle region may comprise about 42 bases in length.
  • 3' of the handle region of the sgRNA is a terminator region.
  • the terminator region is a S. pyogenes terminator region.
  • the terminator region comprises at about 40 bases in length.
  • the terminator region comprises about 10 to 50, 20 to 60, or 30 to 55 bases in length.
  • Design schemes for gRNA sequences described herein may comprise inclusion of a DNA dependent RNA polymerase promoter region 5' upstream of DNA encoding for the gRNA sequence.
  • Exemplary DNA dependent RNA polymerase promoter regions include, without limitation a T3 and a T7 RNA polymerase promoter sequence.
  • FIG. 6A illustrates an arrangement where a T7 promoter region is 5' upstream of a gRNA and the resultant gRNA transcribed is produced wherein the gRNA includes hairpins.
  • a gRNA is designed to lack a sequence that forms a hairpin secondary structure, FIG. 6B. The hairpin secondary structure may be lacking in the Cas9 handle and/or the terminator region.
  • libraries for directing a genomic sequence editing enzyme (e.g., Cas9) to a particular target nucleic acid sequence.
  • libraries comprises oligonucleic acid sequences that encode sequences for dgRNAs.
  • the libraries comprise nucleic acids, wherein each nucleic acid synthesized is a DNA sequence that encodes for a dgRNA sequence as a transcription product.
  • the libraries comprise nucleic acids, wherein each nucleic acid synthesized is a RNA sequence and the dgRNA itself is synthesized.
  • libraries of dgRNAs comprise oligonucleic acid sequences for crRNA and tracrRNA that are synthesized as separate oligonucleic acids.
  • the oligonucleic acid nucleic acids encode for crRNA and tracrRNA separately.
  • the oligonucleic acid nucleic acids encode for single sequence that when transcribed result in a separate crRNA sequence and a separate tracrRNA sequence. Exemplary sequences for crRNA and tracrRNA are seen in Table 1.
  • gRNA libraries described herein may be used for in vitro screening and analysis. An illustration of such an arrangement is depicted in FIG. 7, where a target double-stranded DNA sequence is incubated with a gRNA sequence and Cas9 enzyme. The mixture results in a double strand DNA break. The DNA break may result in a measureable change in the function or expression of a genomic element.
  • gRNAs described herein, or DNA encoding for gRNAs may be added to cells via various methods known in the art, including, without limitation, transfection, transduction, or electroporation.
  • gRNA libraries described herein are used for in vivo or ex vivo screening and analysis.
  • Cells for screening include primary cells taken from living subjects or cell lines.
  • Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants).
  • Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect.
  • gRNA libraries described herein may also be delivered to a multicellular organism.
  • Exemplary multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.
  • libraries comprising nucleic acids for nuclease targeting of a particular target nucleic acid sequence.
  • libraries described herein comprise synthesized nucleic acids, wherein the nucleic acids is DNA, RNA, any analogs, or derivatives thereof.
  • the target nucleic acid sequence comprises DNA, RNA, any analogs, or derivatives thereof.
  • the nuclease cleaves the target nucleic acid sequence.
  • the nuclease binds the target nucleic acid but does not cleave it.
  • nucleases include, but are not limited to, Transcription Activator-Like Effector Nuclease (TALEN), zinc finger nuclease (ZFN), meganuclease, Argonaute, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein.
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN zinc finger nuclease
  • meganuclease Argonaute
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • a model system for targeted gene editing comprises a Cas9-based approach.
  • Cas9 When expressed or transferred into cells alongside a gRNA, Cas9 allows for the targeted introduction or deletion of genetic information via a complex with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequence of mRNA.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • a Cas9 complex as illustrated in FIGS. 1A- 1B, includes a Cas9 protein, engaged with a guide RNA ("gRNA”) transcript.
  • the i llustrated gRNA contains a target sequence region, a PAM region, and a hairpin region.
  • a gRNA shepherds the Cas9 enzyme to a specific stretch of DNA.
  • gRNA depicted is a sgRNA (single stranded guide RNA)
  • the complex may be formed with a dgRNA (dual stranded guide RNA).
  • Cas9 then cleaves the DNA to disable or repair a gene.
  • dCas9 a disabled or "dead" Cas9
  • dCas9 no longer has a splicing function but, with the addition of another enzymatic activity, performs a different target molecule modifying function.
  • tethering a cytidine deaminase to dCas9 converts a C-G DNA base pair into T-A base pair.
  • a different enzyme tethered to the dCas9 results in changing the base C into a T, or a G to an A in a target DNA.
  • the dCas9 process can be modified by fusion of transcription factors to block or activate RNA polymerase activity, resulting in turning off (CRISPRi) or turning on (CRISPRa) gene transcription and therefore regulate gene expression.
  • the dCas9 process is modified by fusion with a transcriptional repressor.
  • the dCas9 process is modified by fusion with a transcriptional activator. In some instances, the dCas9 process is modified by fusion with a plurality of transcriptional repressors or transcriptional activators. In alternative arrangements, a gRNA has multiple sites for cleavage, resulting in a gRNA having multiple regions for gene editing. In the case of Cas9n, or "nicking Cas9,” either the RuvC or UNH cleavage domain is modified to be inactive. This inactivation leaves Cas9 only able to produce only a stranded break in the DNA (a nick), not a double stranded break.
  • two Cas9n enzymes are used to produce the double stranded break. As they can recognize both the upstream and downstream regions of the cut site, off target effects are ablated.
  • a modified Cas9 enzyme instead of using dual Cas9n proteins to generate the off-target effect-free Cas9 cut, a modified Cas9 enzyme has relaxed binding target specificity stringency to allow for less than perfect matches prior to enzymatic activity.
  • the dCas9 process is modified by fusion with a label or tag for detecting a target nucleic acid.
  • the label is a fluorescent marker (e.g., GFP) for detecting the target nucleic acid.
  • the dCas9 is fused to an epitope tag and is used for purification of the target nucleic acid specified by a gRNA.
  • libraries comprising nucleic acids for directing a nuclease to a particular target nucleic acid sequence.
  • the target nucleic acid sequence comprises DNA.
  • the target nucleic acid sequence comprises RNA.
  • libraries comprising nucleic acids for directing C2c2 are generated for targeting a RNA sequence.
  • the DNA or RNA is single stranded or double stranded.
  • libraries comprising nucleic acids for nuclease targeting of a particular target nucleic acid sequence, wherein the nuclease is from a species of, but not limited to, Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,
  • Neisseria Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium,
  • Lachnospiraceae Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Desulfurococcus, Opitutaceae, Tuberibacillus, Bacillus,
  • nucleases Chlamydomonas, Thermus, Pyrococcus, Mycoplasma, or Acidaminococcus.
  • Exemplary nucleases are listed in Table 2A.
  • gRNAs described herein may bind to the terminator sequence of a nuclease from any of the species listed above, or nucleases from additional species where the enzyme allows for genome editing functions.
  • Exemplary terminator sequences include, without limitation, those listed in Table 2B.
  • Exemplary PAM sequences include, without limitation, those listed in Table 2C
  • libraries comprising nucleic acids for targeting one or more nuclease(s) to a particular nucleic acid sequence.
  • the nuclease is at least one of TALEN, ZFN, meganuclease, Argonaute, and Cas protein.
  • more than one nuclease can be multiplexed to generate large genomic deletions, modify multiple sequences at once, or be used in conjunction with other enzymes such as a nickase.
  • the number of nucleases is at least 2 nucleases for the target nucleic acid sequence.
  • the number of nucleases is in a range of about 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 nucleases for the target nucleic acid sequence.
  • a nickase is an enzyme that generates a single stranded break in a nucleic acid sequence.
  • the synthesized nucleic acids are DNA, RNA, any analogs, or derivatives thereof.
  • the particular nucleic acid sequence comprises DNA, RNA, any analogs, or derivatives thereof.
  • the nickase cleaves the particular nucleic acid sequence. In some instances, the nickase binds the particular nucleic acid but does not cleave it. In some instances, the nickase is an altered nuclease, wherein the nuclease is TALEN, ZFN, meganuclease, Argonaute, or Cas protein. In some instances, the nickase is generated by altering a nuclease domain of TALEN, ZFN, meganuclease, Argonaute, or Cas protein. In some instances, the nickase is generated by altering the nuclease domain of Cas9.
  • libraries comprise nucleic acids for one or more nickase(s) targeting of a particular nucleic acid sequence.
  • the number of nickases is at least 2 nickases for the particular nucleic acid sequence.
  • the number of nickases is in a range of about 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 nickases for the particular nucleic acid sequence.
  • libraries comprise nucleic acids for directing one or more nickase and one or more nuclease to the particular nucleic acid sequence.
  • nuclease is at least one of TALEN, ZFN, meganuclease, Argonaute, and Cas protein.
  • nuclease is a chimeric nuclease that provides for a modification of the particular nucleic acid sequence other than cleavage.
  • the chimeric nuclease results in methylation, demethylation, polyadenylation, deadenylation, deamination, or polyuridinylation.
  • TALEN Transcription Activator-Like Effector Nuclease
  • methods for synthesizing nucleic acid libraries comprising nucleic acids for Transcription Activator-Like Effector Nuclease (TALEN) targeting of a particular nucleic acid sequence.
  • TALENs are a class of engineered sequence-specific nucleases that can be used to induce double-strand breaks at specific target sequences.
  • TALENs can be generated by fusing transcription activator- like (TAL) effector DNA-binding domain, or a functional part thereof, to the catalytic domain of a nuclease.
  • the TAL effector DNA binding domain comprises a series of TAL repeats, which are generally highly conserved 33 or 34 amino acid sequence segments that each comprise a highly variable 12th and 13th amino acid known as the repeat variable diresidue (RVD). Each RVD can recognize and bind to a specific nucleotide. Thus, a TAL effector binding domain can be engineered to recognize a specific sequence of nucleotides by combining TAL repeats comprising the appropriate RVDs. [0096] Provided herein are methods for synthesizing a nucleic acid library comprising non- identical nucleic acids that encode for a TAL effector DNA-binding domain.
  • the TAL effector DNA-binding domain is designed to recognize a particular target nucleic acid sequence and induce double-stranded breaks at a particular site.
  • the TAL effector DNA-binding domain comprises a number of TAL repeats that are designed to recognize and bind to a particular nucleic acid sequence.
  • the number of TAL repeats is at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more TAL repeats.
  • a nucleic acid library comprising non-identical nucleic acids encoding for TAL effector DNA-binding domain are synthesized.
  • the nucleic acid library as described herein that when translated encodes for a protein library.
  • the nucleic acid library is expressed in cells and a protein library is generated.
  • the synthesized nucleic acids libraries are inserted into expression vectors.
  • the synthesized nucleic acids libraries are inserted into expression vectors and expressed in cells.
  • Nucleic acid libraries comprising nucleic acids that encode for a TAL effector DNA- binding domain generated by methods described herein can be used for generating a TALEN. In some instances, this is accomplished by mixing the TAL effector binding domain library that is cloned and expressed in vectors with a nuclease.
  • nucleases include, but are not limited to, Acil, Acul, Alwl, Bbvl, Bed, BceAI, BciVI, BfuAI, BmgBI, Bmrl, Bpml, BpuEI, Bsal, BsmAI, BsmFI, BseRI, BspCNI, BsrI, Bsgl, BsmI, BspMI, BsrBI, BsrDI, BtgZI, BtsI, BtsCI, Earl, Ecil, Fokl, Hgal, HphI, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, SfaNI, BbvCI, BpulOI, BspQI, Sapl, Bael, BsaXI, or CspCI.
  • ligases included, but are not limited to, E. coli ligase, T4 ligase, mammalian ligases (e.g., DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV), thermostable ligases, and fast ligases.
  • TALENs generated by methods described herein can be inserted into expression vectors. In some instances, TALENs are inserted into expression vectors and expressed in cells.
  • TAL effector DNA-binding domain library comprising non-identical nucleic acid sequences for a gene in a genome of a prokaryotic or eukaryotic organism.
  • the TAL effector DNA-binding domain library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical nucleic acid sequences for a gene for at least 5% of the genome.
  • the TAL effector DNA-binding domain library comprise non-identical nucleic acid sequences for one or more genes for at least 5% of the genome.
  • the TAL effector DNA-binding domain library comprises non-identical nucleic acid sequence for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes for at least 5% of the genome. In some instances, the TAL effector DNA-binding domain library comprises non-identical nucleic acid sequence for about 1-100, 2-95, 5-90, 10-85, 15-80, 20-75, 25- 70, 30-65, 35-60, or 40-50 genes for at least 5% of the genome.
  • ZFNs can be generated by fusion of a nuclease with a DNA binding zinc finger domain (ZFD).
  • ZFD can bind to a target nucleic acid sequence through one or more zinc fingers.
  • the ZFD comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc fingers.
  • the ZFD comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc fingers.
  • the ZFD is designed to recognize a particular target nucleic acid sequence and induce double-stranded breaks at a particular site.
  • nucleic acid library comprising nucleic acids that when transcribed and translated encode for a ZFD.
  • nucleic acid library when the nucleic acid library is translated encode for a protein library.
  • the nucleic acid library is expressed in cells and a protein library is generated.
  • the synthesized nucleic acids libraries are inserted into expression vectors.
  • the synthesized nucleic acids libraries are inserted into expression vectors and expressed in cells.
  • Nucleic acid libraries comprising nucleic acids that encode for a ZFD generated by methods described herein can be used for generating a ZFN. In some instances, this is
  • nucleases include, but are not limited to, Acil, Acul, Alwl, Bbvl, Bed, BceAI, BciVI, BfuAI, BmgBI, Bmrl, Bpml, BpuEI, Bsal, BsmAI, BsmFI, BseRI, BspCNI, Bsrl, Bsgl, Bsml, BspMI, BsrBI, BsrDI, BtgZI, Btsl, BtsCI, Earl, Ecil, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, SfaNI, BbvCI, BpulOI, BspQI, Sapl, Bael, BsaXI, or CspCI.
  • mixing occurs by ligation.
  • ligases included, but are not limited to, E. coli ligase, T4 ligase, mammalian ligases (e.g., DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV), thermostable ligases, and fast ligases.
  • ZFNs generated by methods described herein can be inserted into expression vectors. In some instances, ZFNs are inserted into expression vectors and expressed in cells.
  • the ZFD library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical nucleic acid sequences for a gene for at least 5% of the genome.
  • the ZFD library comprise non-identical nucleic acid sequences for one or more genes for at least 5% of the genome.
  • the ZFD library comprises non-identical nucleic acid sequence for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes for at least 5% of the genome.
  • the ZFD library comprises non-identical nucleic acid sequence for about 1-100, 2- 95, 5-90, 10-85, 15-80, 20-75, 25-70, 30-65, 35-60, or 40-50 genes for at least 5% of the genome.
  • meganucleases are enzymes that can recognize and cleave long base pair ⁇ e.g., 12-40 base pairs) DNA targets.
  • meganucleases are engineered to comprise domains of other enzymes to confer specificity for a target nucleic acid sequence.
  • meganucleases are engineered to comprise a TAL effector DNA binding domain.
  • nucleic acid library comprising nucleic acids that when transcribed and translated encode for a binding domain for use with a
  • the nucleic acid library when the nucleic acid library is translated encode for a protein library. In some instances, the nucleic acid library is expressed in cells and a protein library is generated. In some instances, the synthesized nucleic acids libraries are inserted into expression vectors. In some instances, the synthesized nucleic acids libraries are inserted into expression vectors and expressed in cells.
  • Nucleic acid libraries comprising nucleic acids that encode for a domain generated by methods described herein can be used for engineering a meganuclease for targeting a particular nucleic acid sequence. In some instances, this is accomplished by mixing a binding domain library such as a TAL effector binding domain library that is cloned and expressed in vectors with a meganuclease.
  • a binding domain library such as a TAL effector binding domain library that is cloned and expressed in vectors with a meganuclease.
  • Exemplary meganucleases for use with the methods provided herein include, but are not limited to, I-Scel, I- Scell, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ceul, I-CeuAIIP, I-Crel, I-CrepsbIP, I- CrepsbllP, I-CrepsbIIIP, I-CrepsbIVP, I-Tlil, I-Ppol, PI-PspI, F-Scel, F-Scell, F-Suvl, F- Tevl, F-TevII, I-Amal, I-Anil, I-Chul, I-Cmoel, I-Cpal, I-CpaII, I-Csml, I-Cvul, I- CvuAIP, I-Ddil, I-DdiII, I-Dirl, I-D
  • ligases included, but are not limited to, E. coli ligase, T4 ligase, mammalian ligases (e.g., DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV), thermostable ligases, and fast ligases.
  • Engineered meganucleases generated by methods described herein can be inserted into expression vectors. In some instances, the engineered meganucleases are inserted into expression vectors and expressed in cells.
  • the domain library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical nucleic acid sequences for a gene for at least 5% of the genome. In some instances, the domain library comprise non-identical nucleic acid sequences for one or more genes for at least 5% of the genome.
  • the domain library comprises non-identical nucleic acid sequence for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes for at least 5% of the genome. In some instances, the domain library comprises non-identical nucleic acid sequence for about 1-100, 2-95, 5-90, 10-85, 15-80, 20-75, 25- 70, 30-65, 35-60, or 40-50 genes for at least 5% of the genome.
  • Argonautes are a family of RNA or DNA guided nucleases. In some instances, Argonautes use a guide nucleic acid to identify a target nucleic acid. In some instances, the guide nucleic acid is a single guide RNA (sgRNA). In some instances, the guide nucleic acid is a guide DNA (gDNA). Exemplary Argonautes include, but are not limited to, TtAgo, PfAgo, and NgAgo. In some embodiments, the Argonaute is NgAgo.
  • the guide nucleic acid library is a sgRNA library. In some instances, the guide nucleic acid library is a dgRNA library. In some instances, the guide nucleic acid library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical nucleic acid sequences for a gene for at least 5% of the genome. In some instances, the guide nucleic acid library comprise non- identical nucleic acid sequences for one or more genes for at least 5% of the genome.
  • the guide nucleic acid library comprises non-identical nucleic acid sequence for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes for at least 5% of the genome. In some instances, the guide nucleic acid library comprises non-identical nucleic acid sequence for about 1-100, 2-95, 5-90, 10-85, 15-80, 20-75, 25-70, 30-65, 35-60, or 40-50 genes for at least 5% of the genome.
  • the Cas protein is at least one of Cpfl, C2cl, C2c2, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Cs
  • gRNA library comprising non-identical nucleic acid sequences for a gene in a genome of a prokaryotic or eukaryotic organism.
  • the gRNA library comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-identical nucleic acid sequences for a gene for at least 5% of the genome.
  • the gRNA library comprise non-identical nucleic acid sequences for one or more genes for at least 5% of the genome.
  • the gRNA library comprises non-identical nucleic acid sequence for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 genes for at least 5% of the genome.
  • the gRNA library comprises non-identical nucleic acid sequence for about 1-100, 2-95, 5-90, 10-85, 15-80, 20-75, 25-70, 30-65, 35-60, or 40-50 genes for at least 5% of the genome.
  • the gRNA library may encode for sgRNA or dgRNAs.
  • a variant nucleic acid library generated by combination of nucleic acids encoding complete or partial gene sequence with gRNAs and a nuclease, e.g., Cas9 enzyme or Cas9 variant enzyme.
  • the fragments may collectively space the entire region of a gene.
  • the library encodes DNA or RNA.
  • the library encodes for a single gene or for up to an entire genome. For example, a gRNA library encoding for 5 gRNAs per a gene for a genome comprising about 20,000 genes would result in about 100,000 gRNAs.
  • Such a library can be used to selectively silence or modify a single gene, a pathway of genes, or all genes in a single genome.
  • gRNAs lack a homology sequence and random end joining occurs. Such a process results in non-homologous end joining ("NHEJ").
  • NHEJ non-homologous end joining
  • an insertion, a deletion, a frameshift, or single base swapping occurs. See FIG. IB.
  • Synthesized libraries described herein may be used for application in CRISPR-Cas9 functions, wherein the gRNA sequence generated is used to disrupt expression of or alter the expression product sequence of a target DNA sequence in a cell or in a mixture comprising a target DNA and Cas9 enzyme.
  • each variant encodes for a codon resulting in a different amino acid during translation. Table 3 provides a listing of each codon possible (and the representative amino acid) for a variant site.
  • nuclease is TALEN, ZFN, or an engineered meganuclease.
  • methods for synthesis of a variant nucleic acid library generated by combination of nucleic acids encoding complete or partial gene sequence with guide nucleic acids such as sgRNAs with a nuclease, wherein the nuclease is Argonaute or a Cas protein are provided herein.
  • Synthesized libraries described herein may be used for application in nuclease functions, wherein the nucleic acid sequence generated is used to disrupt expression of or alter the expression product sequence of a target DNA sequence in a cell or in a mixture comprising a target DNA and a nuclease.
  • each variant encodes for a codon resulting in a different amino acid during translation.
  • Variant nucleic acid libraries as described herein comprise sgRNAs or dgRNAs for varying a target nucleic acid sequence encoding in at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes.
  • each variant encodes for a codon resulting in a different amino acid of a protein domain.
  • the protein domain is a conserved domain or catalytic domain.
  • the protein domain is, but not limited to, a kinase domain, an ATP -binding domain, a GTP -binding domain, a guanine nucleotide exchange factor (GEF) domain, a GTPase activating protein (GAP) domain, a hydrolase domain, an endonuclease domain, an exonuclease domain, a protease domain, a phosphatase domain, a phospholipase domain, a pleckstrin homology domain, a Src homology domain, and a ubiquitin- binding domain.
  • the variant nucleic acid libraries comprise sgRNAs or dgRNAs for targeting a nucleic acid sequence that encodes for variation in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 protein domains.
  • the variants encode for amino acids for a protein with particular activity.
  • the variants encode for a protein that comprises methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreducta
  • a library of gRNA is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription (in vivo or in vitro) to generate gRNA), wherein the library comprises a plurality of gRNA molecules per a gene.
  • the gRNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gRNAs per a gene.
  • the gRNA library is mixed with a Cas9 enzyme and a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • replacement sequences which comprise a homology sequence and a variant nucleic acid sequence such that variation is introduced into target DNA strands.
  • the resultant target DNA library will comprise a plurality of variant DNA sequences. In some instances, variation introduces a deletion, frame shift, or insertion into target DNA sequence.
  • the variant DNA sequences result in variation for at least one codon per a gene or gene fragment.
  • a portion of a gene is inserted into the target DNA or, alternatively, a portion of a target DNA sequence (i.e. a fragment of a gene or an entire gene) is removed from the target DNA.
  • the variant DNA sequences result in variation for at least one transcription regulatory sequence, e.g., a promoter, UTR, or terminator sequence, associated with gene or gene fragment.
  • nuclease cleavage and homologous recombination are incorporated to generate variety in a target DNA library, wherein the nuclease is a TALEN, a ZFN, a meganuclease, a Cas, or an Argonaute.
  • the nuclease is TALEN
  • a library of TAL effector DNA-binding domains is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription and translation (in vivo or in vitro)), wherein the library comprises a plurality of TAL effector DNA- binding domain molecules per a gene.
  • the TAL effector DNA-binding domain library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAL effector DNA-binding domain molecules per a gene.
  • the TAL effector DNA-binding domain library can then be mixed with a nuclease enzyme to generate a TALEN.
  • the TALEN is combined with a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • replacement sequences which comprise a homology sequence and a variant nucleic acid sequence such that variation is introduced into target DNA strands.
  • the resultant target DNA library will comprise a plurality of variant DNA sequences.
  • variation introduces a deletion, frame shift, or insertion into target DNA sequence.
  • the variant DNA sequences result in variation for at least one codon per a gene or gene fragment.
  • a portion of a gene is inserted into the target DNA or, alternatively, a portion of a target DNA sequence (i.e. a fragment of a gene or an entire gene) is removed from the target DNA.
  • the variant DNA sequences result in variation for at least one transcription regulatory sequence, e.g., a promoter, UTR, or terminator sequence, associated with gene or gene fragment.
  • modified Cas9 enzymes are incorporated to generate a variant target DNA library.
  • a library of gRNA is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription to generate gRNA), wherein the library comprises a plurality of gRNA molecules per a gene.
  • the gRNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gRNAs per a gene.
  • the gRNA library is mixed with a modified Cas9 enzyme and a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • the modified Cas9 enzyme has tethered to it another enzyme with nucleic acid sequence modification capabilities.
  • An exemplary modified Cas9 enzymes includes dCas9 process in which a disabled or "dead” Cas9 ("dCas9”) no longer has a splicing function but, with the addition of another enzymatic activity, performs a different target molecule modifying function.
  • tethering a cytidine deaminase to dCas9 converts a C-G DNA base pair into T-A base pair.
  • a different enzyme tethered to the dCas9 results in changing the base C into a T, or a G to an A in a target DNA.
  • the resultant target DNA library comprises a plurality of variant target DNA sequences.
  • variation introduces a deletion, frame shift, or insertion into target DNA sequence.
  • the variant DNA sequences result in variation for at least one codon per a gene or gene fragment.
  • the variant DNA sequences result in variation for at least one transcription regulatory sequence, e.g., a promoter, UTR, or terminator sequence, associated with gene or gene fragment.
  • nuclease is TALEN.
  • a TAL effector DNA binding domain library is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription and translation to generate the TAL effector DNA binding domain library), wherein the library comprises a plurality of non-identical nucleic acid sequences per a gene.
  • the TAL effector DNA binding domain library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-identical nucleic acid sequences per a gene.
  • the TAL effector DNA-binding domain library can then be mixed with a nuclease enzyme to generate a TALEN.
  • the TALEN is then mixed with a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • the nuclease is ZFN.
  • a ZFD library is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription and translation to generate the ZFD library), wherein the library comprises a plurality of non-identical nucleic acid sequences per a gene.
  • the ZFD library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-identical nucleic acid sequences per a gene.
  • the ZFD library can then be mixed with a nuclease enzyme to generate a ZFN.
  • the ZFN is then mixed with a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • the nuclease is a meganuclease.
  • a binding domain library such as a TAL effector DNA binding domain library for targeting the meganuclease to a particular nucleic acid sequence is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription to generate the binding domain library), wherein the binding domain library comprises a plurality of non-identical nucleic acid sequences per a gene.
  • the binding domain library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non- identical nucleic acid sequences per a gene.
  • the binding domain library can then be mixed a meganuclease enzyme to generate an engineered meganuclease.
  • the engineered meganuclease is then mixed with a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • the nuclease is Argonaute.
  • a guide nucleic acid library (gRNA or gDNA) is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription to generate the guide nucleic acid library), wherein the guide nucleic acid library comprises a plurality of non-identical nucleic acid sequences per a gene.
  • the guide nucleic acid library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non- identical nucleic acid sequences per a gene.
  • the guide nucleic acid library is mixed with a modified Argonaute enzyme and a target DNA library, where the target DNA library comprises nucleic acid sequence encoding for at least one gene fragment or at least one gene.
  • the target DNA library may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or gene fragments.
  • the target DNA library comprises sequence from multiple genes in a pathway or from all genes in an organism.
  • the modified nuclease enzyme has tethered to it another enzyme with nucleic acid sequence modification capabilities.
  • exemplary modification capabilities include, but are not limited to, methylation, demethylation, polyadenylation, deadenylation, deamination, and polyuridinylation.
  • a target DNA library comprising a plurality of variant target DNA sequences results in variation.
  • variation introduces a deletion, frame shift, or insertion into target DNA sequence.
  • the variant DNA sequences result in variation for at least one codon per a gene or gene fragment.
  • the variant DNA sequences result in variation for at least one transcription regulatory sequence, e.g., a promoter, UTR, or terminator sequence, associated with gene or gene fragment.
  • nucleic acid library is a gRNA library described herein.
  • nucleic acid library is a DNA library described herein, that when transcribed results in transcription of gRNA sequences.
  • a non-limiting exemplary list of model organisms is provided in Table 4.
  • a library of gRNAs is synthesized (either by de novo synthesis of RNA or de novo synthesis of DNA followed by transcription to generate gRNAs), wherein the library comprises a plurality of gRNA molecules per a gene.
  • a library described herein may comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gRNAs per a gene.
  • the nucleic acids within a de novo synthesized library encode sequences for at least or about 3 non-identical gRNAs per a single gene. In some instances, the nucleic acids encode sequences in a range of about 1 to about 10 non- identical gRNAs per a single gene.
  • the nucleic acids encode sequences for at least or about 1 non-identical gRNAs per a single gene. In some instances, the nucleic acids encode sequences for at most 10 non-identical gRNAs per a single gene. In some instances, the nucleic acids encode sequences for 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 non- identical gRNAs per a single gene. In some instances, the gRNAs are sgRNAs. In some instances, the gRNAs are dgRNAs. [00136] In some instances, a gRNA library described herein comprises one or more non- identical gRNAs per a gene of an organism.
  • the gRNA library comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-identical gRNAs per a gene for the organism.
  • exemplary organisms include, without limitation, Arabidopsis thaliana, Caenorhabditis elegans, Canis lupus familiaris, Chlamydomonas reinhardtii, Danio rerio, Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli, Macaca mulatta, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus, Saccharomyces cerevisiae, Sus scrofa, and Homo sapiens.
  • the gRNAs are sgRNAs. In some instances, the gRNAs are dgRNAs. In some cases, the gRNA library comprises non-identical gRNAs for at least or about 5% of the entire genome of the organism. In some cases, the gRNA library comprises non-identical gRNAs for about 5% to about 100% of the entire genome of the organism. In some instances, the gRNA library comprises non-identical gRNAs for at least or about 80% of the entire genome of the organism. In some instances, the sgRNA library comprises non-identical gRNAs for at least or about 90% of the entire genome of the organism.
  • the gRNA library comprises non-identical gRNAs for at least or about 95% of the entire genome of the organism. In some cases, the gRNA library comprises non-identical gRNAs for at least or about 100% of the entire genome of the organism.
  • the gRNA library comprises non-identical gRNAs for about 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 80%, 5% to 90%, 5% to 95%, 5% to 100%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 100%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 40% to 90%, 40% to 95%, 40% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 60% to 70%, 60% to 80%, 5%
  • the gRNA library comprises sequences from multiple genes in a pathway or from all genes in an organism.
  • the number of gRNAs may comprise at least 2X, 3X, 5X, or 10X per a gene in an organism listed in Table 4.
  • the gRNA library targets at least one of a gene, a group of genes (e.g., 3-10 genes), a pathway (e.g., 10-100 genes), or a chassis (e.g., 100-1000 genes).
  • Described herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from oligonucleic acid synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform.
  • Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of 100 to 1,000 compared to traditional synthesis methods, with production of up to approximately 1,000,000 oligonucleic acids in a single highly-parallelized run.
  • a single silicon plate described herein provides for synthesis of about 6100 non- identical oligonucleic acids.
  • each of the non-identical oligonucleic acids is located within a cluster.
  • a cluster may comprise 50 to 500 non-identical oligonucleic acids.
  • DNA libraries encoding for gRNA libraries described herein have an error rate of less than 1 :500 when compared to predetermined sequences for the DNAs.
  • de novo oligonucleic acids libraries disclosed herein have an aggregated error rate of less than 1 :500, 1 : 1000, 1 : 1500, 1 :2000, 1 :3000, 1 :5000 or less when compared to predetermined sequences for the DNAs.
  • the aggregate error rate is less than 1 : 1000 when compared to predetermined sequences for the DNAs.
  • the error rate may be an aggregate error rate or an average error rate.
  • RNA libraries encoding for gRNA libraries described herein have an error rate of less than 1 :500 when compared to predetermined sequences for the RNAs.
  • de novo oligonucleic acids libraries disclosed herein have an aggregated error rate of less than 1 :500, 1 : 1000, 1 : 1500, 1 :2000, 1 :3000, 1 :5000, 1 : 10,000 or less when compared to
  • the aggregate error rate is less than 1 : 1000 when compared to predetermined sequences for the RNAs.
  • substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of oligonucleic acids.
  • locus refers to a discrete region on a structure which provides support for oligonucleic acids encoding for a single predetermined sequence to extend from the surface.
  • a locus is on a two dimensional surface, e.g., a substantially planar surface.
  • a locus is on a three-dimensional surface, e.g., a well, microwell, channel, or post.
  • a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for oligonucleic acid synthesis, or preferably, a population of identical nucleotides for synthesis of a population of oligonucleic acids.
  • oligonucleic acid refers to a population of oligonucleic acids encoding for the same nucleic acid sequence.
  • a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for oligonucleic acids synthesized within a library using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often.
  • a substrate comprises a surface that supports the synthesis of a plurality of oligonucleic acids having different predetermined sequences at addressable locations on a common support.
  • a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;
  • the substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000;
  • the substrate provides a surface environment for the growth of oligonucleic acids having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.
  • oligonucleic acids are synthesized on distinct loci of a substrate, wherein each locus supports the synthesis of a population of oligonucleic acids. In some cases, each locus supports the synthesis of a population of oligonucleic acids having a different sequence than a population of oligonucleic acids grown on another locus. In some embodiments, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.
  • a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1, 100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000;
  • a substrate comprises about 10,000 distinct loci.
  • the amount of loci within a single cluster is varied in different embodiments.
  • each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci.
  • each cluster includes about 50-500 loci.
  • each cluster includes about 100-200 loci.
  • each cluster includes about 100-150 loci.
  • each cluster includes about 109, 121, 130 or 137 loci.
  • each cluster includes about 19, 20, 61, 64 or more loci.
  • the silicon plate includes about 1-10, 1-50, or 50-500 clusters. In some instances, the silicon plate includes more than about 50, 100, 250, 500, 2500, 5000, 6000, 6150, 10000 or more clusters. In some instances, each cluster includes 121 loci. In some instances, each cluster includes about 50-500, 50-200, 100-150 loci. In some instances, each cluster includes at least about 50, 100, 150, 200, 500, 1000 or more loci. In some instances, a single plate includes 100, 500, 10000, 20000, 30000, 50000, 100000, 500000, 700000, 1000000 or more loci.
  • the number of distinct oligonucleic acids synthesized on a substrate is dependent on the number of distinct loci available in the substrate.
  • the density of loci within a cluster of a substrate is at least or about 1 locus per mm 2 ,
  • loci per mm 2 10 loci per mm 2 , 25 loci per mm 2 , 50 loci per mm 2 , 65 loci per mm 2 , 75 loci per mm 2 , 100 loci per mm 2 , 130 loci per mm 2 , 150 loci per mm 2 , 175 loci per mm 2 , 200 loci per mm 2 , 300 loci per mm 2 , 400 loci per mm 2 , 500 loci per mm 2 , 1,000 loci per mm 2 or more.
  • a substrate comprises from about 10 loci per mm 2 to about 500 mm 2 , from about 25 loci per mm 2 to about 400 mm 2 , from about 50 loci per mm 2 to about 500 mm 2 , from about 100 loci per mm 2 to about 500 mm 2 , from about 150 loci per mm 2 to about 500 mm 2 , from about 10 loci per mm 2 to about 250 mm 2 , from about 50 loci per mm 2 to about 250 mm 2 , from about 10 loci per mm 2 to about 200 mm 2 , or from about 50 loci per mm 2 to about 200 mm 2 .
  • the distance between the centers of two adjacent loci within a cluster is from about 10 um to about 500 um, from about 10 um to about 200 um, or from about 10 um to about 100 um. In some cases, the distance between two centers of adjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um or 100 um. In some cases, the distance between the centers of two adjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
  • each loci has a width of about 0.5 um, 1 um, 2 um, 3 um, 4 um, 5 um, 6 um, 7 um, 8 um, 9 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um or 100 um. In some cases, the each loci is has a width of about 0.5 um to 100 um, about 0.5 um to 50 um, about 10 um to 75 um, or about 0.5 um to 50 um.
  • the density of clusters within a substrate is at least or about 1 cluster per 100 mm 2 , 1 cluster per 10 mm 2 , 1 cluster per 5 mm 2 , 1 cluster per 4 mm 2 , 1 cluster per 3 mm 2 , 1 cluster per 2 mm 2 , 1 cluster per 1 mm 2 , 2 clusters per 1 mm 2 , 3 clusters per 1 mm 2 , 4 clusters per 1 mm 2 , 5 clusters per 1 mm 2 , 10 clusters per 1 mm 2 , 50 clusters per 1 mm 2 or more.
  • a substrate comprises from about 1 cluster per 10 mm 2 to about 10 clusters per 1 mm 2 .
  • the distance between the centers of two adjacent clusters is less than about 50 um, 100 um, 200 um, 500 um, 1000 um, or 2000 um or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50 um and about 100 um, between about 50 um and about 200 um, between about 50 um and about 300 um, between about 50 um and about 500 um, and between about 100 um to about 2000 um.
  • the distance between the centers of two adjacent clusters is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about 0.5 mm and about 5 mm, or between about 0.5 mm and about 2 mm.
  • each cluster has a cross section of about 0.5 to 2 mm, about 0.5 to 1 mm, or about 1 to 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.
  • a substrate is about the size of a standard 96 well plate, for example between about 100 and 200 mm by between about 50 and 150 mm. In some embodiments,
  • a substrate has a diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm.
  • the diameter of a substrate is between about 25 mm and 1000 mm, between about 25 mm and about 800 mm, between about 25 mm and about 600 mm, between about 25 mm and about 500 mm, between about 25 mm and about 400 mm, between about 25 mm and about 300 mm, or between about 25 mm and about 200.
  • substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm.
  • a substrate has a planar surface area of at least about 100 mm 2 ; 200 mm 2 ; 500 mm 2 ; 1,000 mm 2 ; 2,000 mm 2 ; 5,000 mm 2 ; 10,000 mm 2 ; 12,000 mm 2 ; 15,000 mm 2 ; 20,000 mm 2 ; 30,000 mm 2 ; 40,000 mm 2 ;
  • the thickness of a substrate is between about 50 mm and about 2000 mm, between about 50 mm and about 1000 mm, between about 100 mm and about 1000 mm, between about 200 mm and about 1000 mm, or between about 250 mm and about 1000 mm.
  • substrate thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm.
  • the thickness of a substrate varies with diameter and depends on the composition of the substrate. For example, a substrate comprising materials other than silicon has a different thickness than a silicon substrate of the same diameter. Substrate thickness may be determined by the mechanical strength of the material used and the substrate must be thick enough to support its own weight without cracking during handling.
  • Substrates, devices and reactors provided herein are fabricated from any variety of materials suitable for the methods and compositions described herein.
  • substrate materials are fabricated to exhibit a low level of nucleotide binding.
  • substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding.
  • substrate materials are transparent to visible and/or UV light.
  • substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate.
  • conductive materials are connected to an electric ground.
  • the substrate is heat conductive or insulated.
  • a substrate comprises flexible materials.
  • Flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, polypropylene, and the like.
  • a substrate comprises rigid materials. Rigid materials include, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example,
  • a substrate is fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof.
  • a substrate is manufactured with a combination of materials listed herein or any other suitable material known in the art.
  • a substrate comprises raised and/or lowered features.
  • One benefit of having such features is an increase in surface area to support oligonucleic acid synthesis.
  • a substrate having raised and/or lowered features is referred to as a three- dimensional substrate.
  • a three-dimensional substrate comprises one or more channels.
  • one or more loci comprise a channel.
  • the channels are accessible to reagent deposition via a deposition device such as an oligonucleic acid synthesizer.
  • reagents and/or fluids collect in a larger well in fluid communication one or more channels.
  • a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster.
  • a library of oligonucleic acids is synthesized in a plurality of loci of a cluster.
  • the structure is configured to allow for controlled flow and mass transfer paths for oligonucleic acid synthesis on a surface.
  • the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during oligonucleic acid synthesis.
  • the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing an oligonucleic acid such that the excluded volume by the growing oligonucleic acid does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the oligonucleic acid.
  • a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.
  • segregation is achieved by physical structure. In some embodiments, segregation is achieved by differential functionalization of the surface generating active and passive regions for oligonucleic acid synthesis. Differential functionalization is also be achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct oligonucleic acid synthesis locations with reagents of the neighboring spots. In some cases, a device, such as an oligonucleic acid synthesizer, is used to deposit reagents to distinct oligonucleic acid synthesis locations.
  • Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of oligonucleic acids (e.g., more than about 10,000) with a low error rate (e.g., less than about 1 :500, 1 : 1000, 1 : 1500, 1 :2,000; 1 :3,000; 1 :5,000; or 1 : 10,000).
  • a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm 2 .
  • a well of a substrate may have the same or different width, height, and/or volume as another well of the substrate.
  • a channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate.
  • the diameter of a cluster or the diameter of a well comprising a cluster, or both is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.05 mm and about 1 mm, between about 0.05 mm and about 0.5 mm, between about 0.05 mm and about 0.1 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about
  • the diameter of a cluster or well or both is less than or about 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm or 0.05 mm. In some embodiments, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some embodiments, the diameter of a cluster or well, or both is about 1.150 mm. In some embodiments, the diameter of a cluster or well, or both is about 0.08 mm.
  • the diameter of a cluster refers to clusters within a two-dimensional or three- dimensional substrate.
  • the height of a well is from about 20 um to about 1000 um, from about 50 um to about 1000 um, from about 100 um to about 1000 um, from about 200 um to about 1000 um, from about 300 um to about 1000 um, from about 400 um to about 1000 um, or from about 500 um to about 1000 um. In some cases, the height of a well is less than about 1000 um, less than about 900 um, less than about 800 um, less than about 700 um, or less than about 600 um.
  • a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is from about 5 um to about 500 um, from about 5 um to about 400 um, from about 5 um to about 300 um, from about 5 um to about 200 um, from about 5 um to about 100 um, from about 5 um to about 50 um, or from about 10 um to about 50 um. In some cases, the height of a channel is less than 100 um, less than 80 um, less than 60 um, less than 40 um or less than 20 um.
  • the diameter of a channel, locus (e.g., in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1 um to about 1000 um, from about 1 um to about 500 um, from about 1 um to about 200 um, from about 1 um to about 100 um, from about 5 um to about 100 um, or from about 10 um to about 100 um, for example, about 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
  • the diameter of a channel, locus, or both channel and locus is less than about 100 um, 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
  • the distance between the center of two adjacent channels, loci, or channels and loci is from about 1 um to about 500 um, from about 1 um to about 200 um, from about 1 um to about 100 um, from about 5 um to about 200 um, from about 5 um to about 100 um, from about 5 um to about 50 um, or from about 5 um to about 30 um, for example, about 20 um.
  • surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface.
  • surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.
  • adhesion promoter facilitates structured patterning of loci on a surface of a substrate.
  • exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride.
  • the adhesion promoter is a chemical with a high surface energy.
  • a second chemical layer is deposited on a surface of a substrate.
  • the second chemical layer has a low surface energy.
  • surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.
  • a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for oligonucleic acid synthesis are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three- dimensional features).
  • a substrate surface is modified with one or more different layers of compounds.
  • modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.
  • Non-limiting polymeric layers include peptides, proteins, nucleic acids or mimetics thereof (e.g., peptide nucleic acids and the like), polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyetheyleneamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and any other suitable compounds described herein or otherwise known in the art.
  • polymers are heteropolymeric.
  • polymers are homopolymeric.
  • polymers comprise functional moieties or are conjugated.
  • resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy.
  • a moiety is chemically inert.
  • a moiety is configured to support a desired chemical reaction, for example, one or more processes in an oligonucleic acid synthesis reaction.
  • the surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface.
  • a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule.
  • the organofunctional alkoxysilane molecule comprises dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, trimethyl-octodecyl-silane, triethyl-octodecyl-silane, or any combination thereof.
  • a substrate surface comprises functionalized with polyethylene/polypropylene (functionalized by gamma irradiation or chromic acid oxidation, and reduction to hydroxyalkyl surface), highly crosslinked polystyrene-divinylbenzene (derivatized by chloromethylation, and aminated to benzylamine functional surface), nylon (the terminal aminohexyl groups are directly reactive), or etched with reduced polytetrafluoroethylene.
  • polyethylene/polypropylene functionalized by gamma irradiation or chromic acid oxidation, and reduction to hydroxyalkyl surface
  • highly crosslinked polystyrene-divinylbenzene derivatized by chloromethylation, and aminated to benzylamine functional surface
  • nylon the terminal aminohexyl groups are directly reactive
  • a substrate surface is functionalized by contact with a
  • organofunctional alkoxysilane molecules A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy.
  • the organofunctional alkoxysilanes are classified according to their organic functions.
  • Non-limiting examples of siloxane functionalizing reagents include hydroxyalkyl siloxanes (silylate surface, functionalizing with diborane and oxidizing the alcohol by hydrogen peroxide), diol
  • (dihydroxyalkyl) siloxanes silate surface, and hydrolyzing to diol
  • aminoalkyl siloxanes aminoalkyl siloxanes (amines require no intermediate functionalizing step)
  • glycidoxysilanes (3-glycidoxypropyl-dimethyl- ethoxysilane, glycidoxy-trimethoxysilane)
  • mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane
  • bicyclohepthenyl-trichlorosilane butyl-aldehydr-trimethoxysilane, or dimeric secondary aminoalkyl siloxanes.
  • Exemplary hydroxyalkyl siloxanes include allyl trichlorochlorosilane turning into 3-hydroxypropyl, or 7-oct-l-enyl trichlorochlorosilane turning into 8-hydroxyoctyl.
  • the diol (dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived (2,3- dihydroxypropyloxy)propyl (GOPS).
  • the aminoalkyl siloxanes include 3-aminopropyl trimethoxysilane turning into 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl- diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane).
  • Exemplary dimeric secondary aminoalkyl siloxanes include bis (3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine.
  • the functionalizing agent comprises 1 1-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3- aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3 -triethoxysilylpropyl)-4- hy droxybutyrami de .
  • oligonucleic acid synthesis comprises coupling a base with phosphoramidite. In some embodiments, oligonucleic acid synthesis comprises coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. In some embodiments, oligonucleic acid synthesis comprises capping of unreacted sites. In some cases, capping is optional. In some embodiments, oligonucleic acid synthesis comprises oxidation. In some embodiments, oligonucleic acid synthesis comprises deblocking or
  • oligonucleic acid synthesis comprises sulfurization. In some cases, oligonucleic acid synthesis comprises either oxidation or sulfurization. In some
  • the substrate is washed, for example, using tetrazole or acetonitrile.
  • Time frames for any one step in a phosphoramidite synthesis method include less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.
  • Oligonucleic acid synthesis using a phosphoramidite method comprises the subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing oligonucleic acid chain for the formation of a phosphite triester linkage.
  • a phosphoramidite building block e.g., nucleoside phosphoramidite
  • Phosphoramidite oligonucleic acid synthesis proceeds in the 3 ' to 5 ' direction.
  • Phosphoramidite oligonucleic acid synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle.
  • each synthesis cycle comprises a coupling step.
  • Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker.
  • the nucleoside phosphoramidite is provided to the substrate activated.
  • the nucleoside phosphoramidite is provided to the substrate with an activator.
  • nucleoside phosphoramidites are provided to the substrate in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides.
  • nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile.
  • the substrate is optionally washed.
  • the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate.
  • an oligonucleic acid synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps.
  • the nucleoside bound to the substrate is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization.
  • a common protecting group is 4,4'-dimethoxytrityl (DMT).
  • phosphoramidite oligonucleic acid synthesis methods optionally comprise a capping step.
  • a capping step the growing oligonucleic acid is treated with a capping agent.
  • a capping step is useful to block unreacted substrate-bound 5' -OH groups after coupling from further chain elongation, preventing the formation of oligonucleic acids with internal base deletions.
  • phosphoramidites activated with lH-tetrazole may react, to a small extent, with the 06 position of guanosine. Without being bound by theory, upon oxidation with I 2 /water, this side product, possibly via 06-N7 migration, may undergo depurination.
  • the apurinic sites may end up being cleaved in the course of the final deprotection of the oligonucleic acid thus reducing the yield of the full-length product.
  • the 06 modifications may be removed by treatment with the capping reagent prior to oxidation with I 2 /water.
  • inclusion of a capping step during oligonucleic acid synthesis decreases the error rate as compared to synthesis without capping.
  • the capping step comprises treating the substrate-bound oligonucleic acid with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the substrate is optionally washed.
  • the substrate bound growing nucleic acid is oxidized.
  • the oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester
  • oxidation of the growing oligonucleic acid is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (l S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO).
  • a capping step is performed following oxidation. A second capping step allows for substrate drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the substrate and growing oligonucleic acid is optionally washed. In some embodiments, the step of oxidation is substituted with a sulfurization step to obtain oligonucleotide
  • phosphorothioates wherein any capping steps can be performed after the sulfurization.
  • Many reagents are capable of the efficient sulfur transfer, including but not limited to 3- (Dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-3-thione, DDTT, 3H-l,2-benzodithiol-3- one 1, 1 -dioxide, also known as Beaucage reagent, and ⁇ , ⁇ , ⁇ ' ⁇ '-Tetraethylthiuram disulfide (TETD).
  • DDTT Dimethylaminomethylidene)amino-3H-l,2,4-dithiazole-3-thione
  • TETD ⁇ , ⁇ , ⁇ ' ⁇ '-Tetraethylthiuram disulfide
  • the protected 5' end of the substrate bound growing oligonucleic acid is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite.
  • the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product.
  • Methods and compositions of the invention described herein provide for controlled deblocking conditions limiting undesired depurination reactions.
  • the substrate bound oligonucleic acid is washed after deblocking. In some cases, efficient washing after deblocking contributes to synthesized oligonucleic acids having a low error rate.
  • Methods for the synthesis of oligonucleic acids typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking.
  • One or more intermediate steps include oxidation or sulfurization.
  • one or more wash steps precede or follow one or all of the steps.
  • Methods for phosphoramidite based oligonucleic acid synthesis comprise a series of chemical steps.
  • one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the substrate of a reagent useful for the step.
  • reagents are cycled by a series of liquid deposition and vacuum drying steps.
  • substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the substrate via the wells and/or channels.
  • Oligonucleic acids synthesized using the methods and/or substrates described herein comprise, in various embodiments, at least about 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 120, 150 or more bases. In some embodiments, at least about 1 pmol, 10 pmol, 20 pmol, 30 pmol, 40 pmol, 50 pmol, 60 pmol, 70 pmol, 80 pmol, 90 pmol, 100 pmol, 150 pmol, 200 pmol, 300 pmol, 400 pmol, 500 pmol, 600 pmol, 700 pmol, 800 pmol, 900 pmol, 1 nmol, 5 nmol, 10 nmol, 100 nmol or more of an oligonucleic acid is synthesized within a locus.
  • Methods for oligonucleic acid synthesis on a surface allow for synthesis at a fast rate.
  • at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized.
  • Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof.
  • libraries of oligonucleic acids are synthesized in parallel on substrate.
  • a substrate comprising about or at least about 100; 1,000; 10,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct oligonucleic acids, wherein oligonucleic acid encoding a distinct sequence is synthesized on a resolved locus.
  • a library of oligonucleic acids are synthesized on a substrate with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.
  • larger nucleic acids assembled from an oligonucleic acid library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.
  • a predetermined library of oligonucleic acids is designed for de novo synthesis.
  • Various suitable methods are known for generating high density oligonucleic acid arrays.
  • a substrate surface layer is provided.
  • chemistry of the surface is altered in order to improve the oligonucleic acid synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids.
  • the surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area.
  • high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.
  • oligonucleic acid arrays are generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel.
  • a deposition device such as an oligonucleic acid synthesizer, is designed to release reagents in a step wise fashion such that multiple oligonucleic acids extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence.
  • oligonucleic acids are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.
  • any of the systems described herein may be operably linked to a computer and may be automated through a computer either locally or remotely.
  • the methods and systems of the invention may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the invention.
  • the computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.
  • the computer system 800 illustrated in FIG. 8 may be understood as a logical apparatus that can read instructions from media 811 and/or a network port 805, which can optionally be connected to server 809 having fixed media 812.
  • the system such as shown in FIG. 8 can include a CPU 801, disk drives 803, optional input devices such as keyboard 815 and/or mouse 816 and optional monitor 807.
  • Data communication can be achieved through the indicated communication medium to a server at a local or a remote location.
  • the communication medium can include any means of transmitting and/or receiving data.
  • the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 822 as illustrated in FIG. 8.
  • FIG. 9 is a block diagram illustrating a first example architecture of a computer system 900 that can be used in connection with example embodiments of the present invention.
  • the example computer system can include a processor 902 for processing instructions.
  • processors include: Intel XeonTM processor, AMD
  • OpteronTM processor Samsung 32-bit RISC ARM 1176JZ(F)-S vl .OTM processor, ARM Cortex- A8 Samsung S5PC100TM processor, ARM Cortex-A8 Apple A4TM processor, Marvell PXA 930TM processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some embodiments, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.
  • a high speed cache 904 can be connected to, or incorporated in, the processor 902 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 902.
  • the processor 902 is connected to a north bridge 906 by a processor bus 908.
  • the north bridge 906 is connected to random access memory (RAM) 910 by a memory bus 912 and manages access to the RAM 910 by the processor 902.
  • the north bridge 906 is also connected to a south bridge 914 by a chipset bus 916.
  • the south bridge 914 is, in turn, connected to a peripheral bus 918.
  • the peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus.
  • system 900 can include an accelerator card 922 attached to the peripheral bus 918.
  • the accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing.
  • FPGAs field programmable gate arrays
  • an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.
  • the system 900 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux,
  • system 900 also includes network interface cards (NICs) 920 and 921 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.
  • NICs network interface cards
  • NAS Network Attached Storage
  • FIG. 10 is a diagram showing a network 1000 with a plurality of computer systems 1002a, and 1002b, a plurality of cell phones and personal data assistants 1002c, and Network Attached Storage (NAS) 1004a, and 1004b.
  • systems 1002a, 1002b, and 1002c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 1004a and 1004b.
  • NAS Network Attached Storage
  • a mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 1002a, and 1002b, and cell phone and personal data assistant systems 1002c.
  • Computer systems 1002a, and 1002b, and cell phone and personal data assistant systems 1002c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 1004a and 1004b.
  • FIG. 10 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various embodiments of the present invention.
  • a blade server can be used to provide parallel processing.
  • Processor blades can be connected through a back plane to provide parallel processing.
  • Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface.
  • processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors.
  • some or all of the processors can use a shared virtual address memory space.
  • FIG. 11 is a block diagram of a multiprocessor computer system using a shared virtual address memory space in accordance with an example embodiment.
  • the system includes a plurality of processors 1102a-f that can access a shared memory subsystem 1104.
  • the system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 1106a-f in the memory subsystem 1104.
  • MAPs programmable hardware memory algorithm processors
  • Each MAP 1106a-f can comprise a memory 1108a-f and one or more field programmable gate arrays (FPGAs) lllOa-f.
  • the MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs lllOa-f for processing in close coordination with a respective processor.
  • the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example embodiments.
  • each MAP is globally accessible by all of the processors for these purposes.
  • each MAP can use Direct Memory Access (DMA) to access an associated memory 1108a-f, allowing it to execute tasks independently of, and asynchronously from, the respective microprocessor 1102a-f.
  • DMA Direct Memory Access
  • a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.
  • the above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example embodiments, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements.
  • all or part of the computer system can be implemented in software or hardware.
  • Any variety of data storage media can be used in connection with example embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.
  • NAS Network Attached Storage
  • the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems.
  • the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 11, system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements.
  • FPGAs field programmable gate arrays
  • SOCs system on chips
  • ASICs application specific integrated circuits
  • the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 922 illustrated in FIG. 9.
  • Example 1 Functionalization of a substrate surface
  • a substrate was functionalized to support the attachment and synthesis of a library of oligonucleic acids.
  • the substrate surface was first wet cleaned using a piranha solution comprising 90% H 2 SO 4 and 10% H 2 0 2 for 20 minutes.
  • the substrate was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N 2 .
  • the substrate was subsequently soaked in H 4 OH (1 : 100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun.
  • the substrate was then plasma cleaned by exposing the substrate surface to 0 2 .
  • a SAMCO PC-300 instrument was used to plasma etch 0 2 at 250 watts for 1 min in downstream mode.
  • the cleaned substrate surface was actively functionalized with a solution comprising N- (3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70 °C, 135 °C vaporizer.
  • the substrate surface was resist coated using a Brewer Science 200X spin coater. SPRTM 3612 photoresist was spin coated on the substrate at 2500 rpm for 40 sec. The substrate was pre-baked for 30 min at 90 °C on a Brewer hot plate. The substrate was subjected to photolithography using a Karl Suss MA6 mask aligner instrument.
  • the substrate was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the substrate soaked in water for 5 min. The substrate was baked for 30 min at 100 °C in the oven, followed by visual inspection for lithography defects using a Nikon L200. A cleaning process was used to remove residual resist using the SAMCO PC-300 instrument to 0 2 plasma etch at 250 watts for 1 min. [00191] The substrate surface was passively functionalized with a 100 ⁇ . solution of perfluorooctyltrichlorosilane mixed with 10 ⁇ . light mineral oil. The substrate was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min.
  • the chamber was vented to air.
  • the substrate was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70 °C with ultrasonication at maximum power (9 on Crest system).
  • the substrate was then soaked for 5 min in 500 mL isopropanol at room temperature with
  • the substrate was dipped in 300 mL of 200 proof ethanol and blown dry with N 2 .
  • the functionalized surface was activated to serve as a support for oligonucleic acid synthesis.
  • EXAMPLE 2 Synthesis of a 50-mer sequence on an oligonucleic acid synthesis device
  • a two dimensional oligonucleic acid synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems "ABI394 DNA Synthesizer").
  • the two- dimensional oligonucleic acid synthesis device was uniformly functionalized with N-(3- TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to synthesize an exemplary oligonucleic acid of 50 bp ("50-mer oligonucleic acid”) using oligonucleic acid synthesis methods described herein.
  • Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02 M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ⁇ 100uL/sec, for acetonitrile (“ACN”) and capping reagents (1 : 1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ⁇ 200uL/sec, and for Deblock (3%
  • EXAMPLE 3 Synthesis of a 100-mer sequence on an oligonucleic acid synthesis device
  • Table 7 summarizes key error characteristics for the sequences obtained from the oligonucleic acid samples from spots 1-10.
  • EXAMPLE 4 sgRNA Design
  • a chimera sgRNA sequence with a variable region at the 5' end was designed for direct sequence specific cleavage by the Cas9 protein. See FIG. 4A.
  • the sgRNA sequence had a base- pairing region of 20 bases for specific DNA binding, which included a seed region of 12 bases.
  • the 5' end of the base-pairing region was designed to be the transcription start site.
  • 3' proximal to the base-pairing region was the dCas9 handled region for Cas9 binding, which was 42 bases in length.
  • 3' proximal to the dCas9 handled region was the S. pyogenes terminator region which was 40 bases in length.
  • the dCas9 handled region and the terminator region each were designed to include sequence that would result in a hairpin structure.
  • sgRNAs were also designed to target the template (T) or nontemplate (NT) DNA strands, FIGS. 5A-5B.
  • sgRNAs designed for targeting the template DNA strand included a base-pairing region of the sgRNA having the same sequence identity as the transcribed sequence.
  • sgRNAs designed for targeting the nontemplate DNA strand included the base-pairing region of the sgRNA that was a reverse-complement of the transcribed sequence.
  • a T7 promoter was designed immediately upstream of variable base-pairing region. See FIGS. 6A-6B.
  • the T7 promoter region was added to enable in vitro production of the sgRNA with T7 polymerase.
  • EXAMPLE 5 Synthesis of DNA encoding for sgRNA - Design and Polymerase Analysis
  • DNA oligonucleic acids were designed as fragments that, when joined, encode for an sgRNA sequence.
  • FIG. 12. The sgRNAs were designed for inclusion of a T7 promoter
  • a sequence for Design 1 1220, Design 2 1222, Design 3 1224, and Design 4 1226 was designed as indicated in Table 8.
  • the sequence for each Design 1, Design 2, Design 3, and Design 4 comprises a T7 promoter, a variable sequence portion, and a constant sequence region (the handle and terminator) (Table 8).
  • the constant sequence region as seen in FIG. 12 comprises a Cas9 handle hairpin comprising base pairing regions 1211, 1213, 1215, 1217, 1223, and 1225, and a terminator hairpin comprising base pairing regions 1219 and 1221.
  • T7 RNA polymerase promoter region should be double stranded for recognition by T7 RNA polymerase.
  • An antisense oligonucleic acid was used for hybridization: 5'- TAATACGACTCACTATAGG- 3' (SEQ ID NO: 18).
  • Table 9 provides a list of primers that were used for analysis of 4 different sets of template and
  • oligonucleic acid designs 3 and 4 resulted in increased DNA yield with all three polymerases.
  • the higher annealing temperature of 60 °C resulted in increased DNA yield.
  • EXAMPLE 6 CRISPR sgRNA Synthesis - Temperature Analysis
  • Amplification product was run on a BioAnalyzer (data not shown) to estimate the yield, and is summarized in Table 15.
  • DNA yield is presented in ng/ul (Table 15).
  • Polymerase 3 provides increased DNA yield and 60 °C annealing temperature resulted in an increased DNA yield.
  • EXAMPLE 7 sgRNA Generation - Structure Free RNA
  • Two assembly oligonucleic acids were designed to generate a modified sgRNA template (120 bp) with T7 promoter sequence and terminator, but without the tracrRNA hairpin containing sequence. See Table 16.
  • RNA product of 80bp was expected to yield an RNA product of 80bp, devoid of secondary structure. Transcription of the amplification product was carried out with an in vitro transcription kit (NEB HiScribe). The reaction mixture was analyzed on a BioAnalyzer. See FIGS. 13A-13B. The modified sgRNA product was cleaner with the structure free design (FIG. 13B) than the sgRNA having the tracrRNA hairpin containing sequence (FIG. 13A).
  • EXAMPLE 8 sgRNA Directed Cas9 Cleavage
  • sgRNA sequences were designed with a T7 promoter region and each with a different recognition sequence for regions of a 720 bp GFP encoding sequence.
  • Each of the sgRNA sequences was assembled from PCR of two oligonucleic acids.
  • the sgRNA backbone and primers are provided in Table 18.
  • Cas9 digests were prepared using GFP amplification product, Cas9 and the transcribed sgRNA. 2 peaks were observed for all three digests, compared to a single peak for the control. (FIGS. 14G-14J). Expected and resultant fragments from Cas9 cleavage using the 3 synthesized sgRNAs are listed Table 20. Table 20.
  • a structure comprising 256 clusters 1505 each comprising 121 loci on a flat silicon plate was manufactured as shown in FIG. 15.
  • An expanded view of a cluster is shown in 1510 with 121 loci.
  • Loci from 240 of the 256 clusters provided an attachment and support for the synthesis of oligonucleic acids having distinct sequences. Oligonucleic acid synthesis was performed by phosphoramidite chemistry using general methods from Example 3.
  • Loci from 16 of the 256 clusters were control clusters.
  • the global distribution of the 29,040 unique oligonucleic acids synthesized (240 non-control clusters x 121 oligonucleic acid populations per cluster) is shown in FIG. 16A.
  • NGS sequencing confirmed 100% representation of designed oligonucleic acids selected for synthesis. Distribution was measured for each cluster, as shown in FIG. 16B. The distribution of unique oligonucleic acids synthesized in 4 representative clusters is shown in FIG. 17. On a global level, all oligonucleic acids the designed for synthesis were present and 99% of the oligonucleic acids had abundance that was within 2x of the mean, indicating high synthesis uniformity. This same observation was consistent on a per-cluster level.
  • the error rate for each oligonucleic acid was determined using an Illumina MiSeq gene sequencer.
  • the error rate distribution for the 29,040 unique oligonucleic acids is shown in FIG. 18A and averages around 1 in 500 bases, with some error rates as low as 1 in 800 bases. Distribution was measured for each cluster, as shown in FIG. 18B.
  • the error rate distribution for unique oligonucleic acids in four representative clusters is shown in FIG. 19.
  • the library of 29,040 unique oligonucleic acids was synthesized in less than 20 hours. Analysis of GC percentage v. oligonucleic acid representation across all of the 29,040 unique oligonucleic acids showed that synthesis was uniform despite GC content (roughly 20% to 85% GC per oligonucleic acid) , FIG. 20.
  • EXAMPLE 10 PCR amplification analysis of de novo synthesized DNA library encoding for sgRNAs
  • oligonucleic acids 100 bases in length of randomized sequences with varying GC content, from 20-80%) GC were designed and synthesized on a structure with a similar arrangement is described in Example 9.
  • the oligonucleic acid population was amplified for either 6 or 20 cycles with a high fidelity DNA polymerase (DNA polymerase 1).
  • the oligonucleic acid population was amplified using two other high-fidelity PCR enzymes for 6, 8, 10, or 15 cycles, to determine whether polymerase selection had an effect on overall sequence representation post-amplification.
  • EXAMPLE 11 Human Epigenetic CRISPR Screen
  • a sgRNA screen was performed to introduce mutations into exons that encode functional domains using CRISPR-Cas9.
  • About 10,000 DNA oligonucleic acids were de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement is described in Example 9.
  • Collectively, the oligonucleic acids had an aggregated error rate of about 1 :500 or lower.
  • Each oligonucleic acid was up to 200 bases in length, and at least 1 fmole per an oligonucleic acid species was generated.
  • the oligonucleic acids were PCR amplified, cloned into vectors, and electroporated into cultured cells for sgRNA transcription. Nucleic acids were isolated from the cells and sequenced, using next generation sequencing.
  • Sequencing results showed highly accurate and uniform library synthesis with minimal bias and high fidelity production of sgRNAs. More reads per guide sequence with minimal sequencing 30% higher recovery of sgRNA with correct sequence for downstream screening compared to competitor pool. See Table 23. Pooled sequencing results showed more reads per guide sequence and a much tighter distribution of reads (4 logs) compared to 6 logs with the array based competitor pool. See FIGS. 24A-24B. Sequencing validation of clones showed 100% sgRNA recovery (FIG. 24A) and higher sequence accuracy compared to a commercially available array-based pool (FIG. 24B). Of the clones that were sequenced, significantly more were recovered with the correct sgRNA sequence. See Table 23.
  • Ave reads per sgRNA in cloned oligo about 256 about 1024
  • EXAMPLE 12 Whole Genome sgRNA Library
  • a DNA library was designed to include DNAs encoding for sgRNAs for generating clones for 101,000 different oligonucleic acids (5 sgRNAs per 20200 gene targets). 101,000 oligonucleic acids were de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement is described in Example 9. The synthesized oligonucleic acids were PCR amplified, digested and cloned into lentiviral vectors, and transformed into cells. Nucleic acids were isolated from the cells and sequenced, using next generation sequencing. Alternatively, the synthesized oligonucleic acids were PCR amplified to form an amplicon-based library and sequenced.
  • a plot of next generation sequencing reads v. number of sgRNAs recovered shows that as the oligonucleic acid pool size increases, the oligonucleic acid population maintained a more uniform tighter distribution of reads across the entire library, with a minimal tail compared to a commercially available array-based reference oligonucleic acid population.
  • FIG. 25 A plot of next generation sequencing reads v. number of sgRNAs recovered shows that as the oligonucleic acid pool size increases, the oligonucleic acid population maintained a more uniform tighter distribution of reads across the entire library, with a minimal tail compared to a commercially available array-based reference oligonucleic acid population.
  • EXAMPLE 13 Design of sgRNA Libraries with Improved Targeting and Activity
  • sgRNA libraries were designed and de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement is described in Example 9.
  • the synthesized oligonucleic acids were PCR amplified, digested and cloned into vectors, and transferred into cells for use for downstream applications including screening and analysis.
  • sgRNA design parameters were compared including libraries characterized by a NAG PAM, a NGG PAM, high activity, low off-target, and filtered.
  • the sgRNA library designed by methods described herein provided for a higher percentage of sgRNAs resulting in at least 2-fold depletion of gene expression, around 16% of sgRNAs, compared to other commercially available gRNA systems.
  • FIG. 26A The sgRNA libraries also provided for a lower percentage of sgRNAs resulting in zero or negative depletion of gene expression, around 17%, compared to other commercially available gRNA systems.
  • FIG. 26B The sgRNA libraries also provided for a lower percentage of sgRNAs resulting in zero or negative depletion of gene expression, around 17%, compared to other commercially available gRNA systems.
  • sgRNA-mediated depletion was assessed for essential gene expression levels as well, where the following genes were targeted by sgRNAs: PCNA, PSMA7, RPP21, and SF3B3.
  • the sgRNA library had a higher percentage sgRNAs depleting essential genes as compared to Comparator 1, Comparator 2, and Comparator 3. See Table 24. Table 24.
  • a DNA library comprising non-identical DNA sequences encoding for sgRNAs was designed for sequence specific cleavage by the C2c2 protein.
  • the library comprised all possible spacer sequences for C2c2 targeting of bacteriophage MS2 genome. Because mature crRNAs of C2c2 from Leptotrichia shahii comprises a maximum spacer length of 28 nucleotides, tiling all possible 28 nucleotide target sites in the bacteriophage genome resulted in a library of about 3500 spacer sequences.
  • Example 9 About 3500 non-identical oligonucleic acids were de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement is described in Example 9. The library of about 3500 sequences were inserted into vectors and transformed into E. coli. E. coli cells were infected with MS2
  • spacer sequences were found to confer resistance. Comparing spacer representation (crRNA frequencies), many spacer sequences exhibited more than 1.25 log 2 -fold enrichment in the three dilutions of MS2 infection whereas no non-targeting spacer sequences were found to be enriched.
  • EXAMPLE 15 sgRNA Library for Zebrafish
  • a DNA library is designed with sequences encoding for about 130,000 sgRNAs. On average, about 5 sgRNAs templates are designed for each zebrafish gene.
  • the oligonucleic acids are de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement as described in Example 9. De novo synthesis produces the 130,000 oligonucleic acids, each extending from a different locus on the surface of a silicon plate.
  • the oligonucleic acids are removed from the plate, amplified by PCR, and cloned into expression vectors.
  • Each template is subject to sequencing.
  • the sgRNA library is injected into zebrafish embryos. Zebrafish are raised to adulthood.sperm are then cryopreserved and screened by sequencing to identify the sequence of germline transmitted insertions and deletions. Following the germline screen, sperm are genotyped by competitive allele-specific PCR.
  • EXAMPLE 16 gRNA Library for Mouse
  • a DNA library is designed with sequences encoding for about 100,000 sgRNAs. On average, about 5 sgRNAs templates are designed per mouse gene.
  • the oligonucleic acids are de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement as described in Example 9.
  • a sgRNA library encoding for the sgRNA sequences is de novo synthesized to generate 100,000 oligonucleic acids. De novo synthesis produces the 100,000 oligonucleic acids, each extending from a different locus on the surface of a silicon plate. The oligonucleic acids are removed from the plate, amplified by PCR, and cloned into vectors. Each template is subject to sequencing.
  • sgRNA on-target efficiency is verified by surveyor nuclease assay or sequencing. sgRNAs are then microinjected in mouse zygotes with a desired genetic background. Alternately, following verification of sgRNA efficiency, sgRNAs are packaged into viral vectors such as adeno-associated viruses (AAVs). sgRNAs are then stereotactically delivered into mice at a desired location.
  • AAVs adeno-associated viruses
  • Expression levels for the preselected target genes are observed in tissue collected from mice.
  • EXAMPLE 17 gRNA library for a Receptor Tyrosine Kinases
  • a DNA oligonucleic acid library is designed with sequences encoding for 5 sgRNAs targeting genes for 58 human receptor tyrosine kinases listed in Table 25, totaling 290 different DNA oligonucleic acids.
  • the oligonucleic acids are de novo synthesized using methods similar to those described in Example 3 on a silicon chip as described in Example 1 on a structure with a similar arrangement as described in Example 9.
  • the oligonucleic acids are removed from the plate, amplified by PCR, cloned into vectors, and transferred into preselected populations of cells.
  • Expression levels for the preselected genes listed in Table 25 are compared in each preselected populations of cells against a control population of cells exposed to a control vector without the kinase-specific sgRNA.
  • EXAMPLE 18 gRNA library for Human Kinome
  • a DNA oligonucleic acid library is designed with sequences encoding for 5 sgRNAs targeting genes for 518 human kinases, totaling 2,590 different DNA oligonucleic acids.
  • the oligonucleic acids are removed from the plate, amplified by PCR, cloned into vectors, and transferred into preselected populations of cells. Expression levels for the preselected 518 genes are compared in each preselected populations of cells against a control population of cells exposed to a control vector without the kinase-specific sgRNA.
  • EXAMPLE 19 gRNA library for Human Phosphatome
  • a DNA oligonucleic acid library is designed with sequences encoding for 5 sgRNAs targeting genes for 200 human phosphatases, totaling 1000 different DNA oligonucleic acids.
  • the oligonucleic acids are removed from the plate, amplified by PCR, cloned into vectors, and transferred into preselected populations of cells. Expression levels for the 200 preselected genes are compared in each preselected populations of cells against a control population of cells exposed to a control vector without the kinase-specific sgRNA.

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