(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19)
`
`\
`W 1d 1 t 11
`t
`1 P
`t
`T
`"r 0,232,223,131, mp" y
`’/
`International Bureau
`(43) International Publication Date /
`20 September 2018 (20.09.2018) WI PO 1 P C T
`
`|||||||||||||| |||||||| III” "III "III II" | II III ||||||||||||||| ||||| |||||||||||||||||||| ||||||||
`(10) International Publication Number
`W0 201 8/1 701 64 A1
`
`(51) International Patent Classification:
`("403 30/06 (2006.01)
`C12N5/071 (2010.01)
`C40B 40/08 (2006.01)
`CIZZV 5/10 (2006.01)
`C40B 50/02 (2006.01)
`C12Q 1/68 (2018.01)
`C40B 50/06 (2006.01)
`A61K 31/711 (2006.01)
`CIZN 15/66 (2006.01)
`(21) International Application Number:
`
`PCT/US2018/022487
`
`(22) International Flhng Date:
`
`14 March 2018 (14.03.2018)
`
`(25) “mg Language:
`(26) Publication Language:
`(30) Priority Data:
`62/471,723
`62/578,326
`
`15 March 2017 (15.03.2017)
`27 October 2017 (27.10.2017)
`
`EnghSh
`English
`
`US
`US
`
`(71) Applicant: TWIST BIOSCIENCE CORPORATION
`[US/US]; 455 Mission Bay Blvd. South, Suite 545, San
`Francisco, California 94158 (US).
`
`(72) Inventors: COX, Anthony; 455 Mission Bay Blvd. South,
`Suite 545, SanFranCisco, California 94158 (US). CHEN,
`Siyuan; 455 Mission Bay Blvd. South, Suite 545, San Fran-
`cisco, California 94158 (US). LEDOGAR, Charles; 455
`Mission Bay Blvd. South, Suite 545, San Francisco, Califor-
`nia 94158 (US). TOPPANI, Dominique; 455 Mission Bay
`Blvd. South, Suite 545, San Francisco, California 94158
`(US).
`
`(74) Agent: HARBURGER, David; WILSON SONSINI
`GOODRICH & ROSATI, 650 Page Mill Road, Palo Alto,
`C' If
`" 94304 US .
`d 1 0mm
`(
`)
`(81) Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GII, GM, GT, IIN,
`
`(54) Title: DE NOVO SYNTHESIZED COMBINATORIAL NUCLEIC ACID LIBRARIES
`
`
`
`Variants: Expected vs Observed
`
`Observed Frequency
`- - Expected Probability
`
`9.00
`
`
`
`
`
`
`
`5.005436
`
`0,005+00
`
`3‘
`4005-06
`L:
`3.50505 5
`EDGE—06 3
`O
`2505-06 5
`K
`v-I
`
`2.00505
`1505-06
`1005—06
`5.00507
`
`7.00
`‘5
`3
`5 5.00
`w
`5.00
`9‘5)
`,_
`0 4.00
`3
`
`3'00
`2-00
`1-00
`0.00
`
`FIG. 22
`
`(57) Abstract: Disclosed herein are methods for the generation of highly accurate nucleic acid libraries encoding for predetermined
`variants of a nucleic acid sequence. The degree of variation may be complete, resulting in a saturated variant library, or less than
`complete, resulting in a non—saturating library of variants. The variant nucleic acid libraries described herein may be designed for
`fiu’ther processing by transcription or translation. The variant nucleic acid libraries described herein may be designed to generate variant
`RNA, DNA and/or protein populations. Further provided herein are method for identifying variant species With increased 01' decreased
`activities, with applications in regulating biological functions and the design of therapeutics for treatment or reduction of disease.
`
`[Continued on nextpage]
`
`
`
`wo2018/170164A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`

`

`WO 2018/170164 A1 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,
`KR, KW, Kz, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`
`— as to applicant’s entitlement to applyfor and be granted a
`patent (Rule 4.17(ii))
`Published:
`
`— with international search report (Art. 21(3))
`— before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`— with sequence listingpart ofdescription (Rule 5.2(a))
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`DE NOVO SYNTHESIZED COMBINATORIAL NUCLEIC ACID LIBRARIES
`
`CROSS-REFERENCE
`
`[0001]
`
`This application claims the benefit of US. Provisional Application No. 62/578,326,
`
`filed on October 27, 2017; and US. Provisional Application No. 62/471,723, filed on March 15,
`
`2017, each of which is incorporated herein by reference in its entirety.
`
`SEQUENCE LISTING
`
`[0002]
`
`The instant application contains a Sequence Listing which has been submitted
`
`electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII
`
`copy, created on March 13, 2018, is named 44854-729760178tht and is 18,419 bytes in size.
`
`BACKGROUND
`
`[0003]
`
`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. In the design phase, the A, C, T and G
`
`nucleotides that constitute DNA are formulated into the various gene sequences that would
`
`comprise the locus or the pathway of interest, with each sequence variant representing a specific
`
`hypothesis that will be tested. These variant gene sequences represent sub sets 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.
`
`[0004]
`
`Many different variants are typically designed for each design-build-test cycle to enable
`
`adequate sampling of sequence space and maximize the probability of an optimized design. Though
`
`straightforward in concept, process bottlenecks around speed, throughput and quality of
`
`conventional synthesis methods dampen the pace at which this cycle advances, extending
`
`development time. The inability to sufficiently explore sequence space due to the high cost of
`
`acutely accurate DNA and the limited throughput of current synthesis technologies remains the
`
`rate-limiting step.
`
`[0005]
`
`Beginning with the build phase, two processes are noteworthy: nucleic acid synthesis
`
`and gene synthesis. Historically, synthesis of different gene variants was accomplished through
`
`molecular cloning. While robust, this approach is not scalable. Early chemical gene synthesis
`
`efforts focused on producing a large number of polynucleotides with overlapping sequence
`
`homology. These were then pooled and subjected to multiple rounds of polymerase chain reaction
`
`(PCR), enabling concatenation of the overlapping polynucleotides into a full length double stranded
`
`gene. A number of factors hinder this method, including time-consuming and labor-intensive
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`construction, requirement of high volumes of phosphoramidites, an expensive raw material, and
`
`production of nanomole amounts of the final product, significantly less than required for
`
`downstream steps, and a large number of separate polynucleotides required one 96 well plate to set
`
`up the synthesis of one gene.
`
`[0006]
`
`Synthesizing of polynucleotides on microarrays provided a significant increase in the
`
`throughput of gene synthesis. A large number of polynucleotides could be synthesized on the
`
`microarray surface, then cleaved off and pooled together. Each polynucleotide destined for a
`
`specific gene contains a unique barcode sequence that enabled that specific subpopulation of
`
`polynucleotides to be depooled and assembled into the gene of interest. In this phase of the process,
`
`each subpool is transferred into one well in a 96 well plate, increasing throughput to 96 genes.
`
`While this is two orders of magnitude higher in throughput than the classical method, it still does
`
`not adequately support the design, build, test cycles that require thousands of sequences at one time
`
`due to a lack of cost efficiency and slow turnaround times.
`
`BRIEF SUMMARY
`
`[0007]
`
`Provided herein are methods of synthesizing a variant nucleic acid library, comprising:
`
`(a) providing predetermined sequences encoding for at least 500 polynucleotide sequences, wherein
`
`the at least 500 polynucleotide sequences have a preselected codon distribution, (b) synthesizing a
`
`plurality of polynucleotides encoding for the at least 500 polynucleotide sequences; (c) assaying an
`
`activity for nucleic acids encoded by or proteins translated based on the plurality of
`
`polynucleotides, and (d) collecting results from the assay in step (0), wherein the collecting
`
`comprises collecting results of predetermined sequences associated with a negative or null result.
`
`Further provided herein are methods of synthesizing a variant nucleic acid library, wherein step ((1)
`
`comprises collecting results for at least 80% of the predetermined sequences. Further provided
`
`herein are methods of synthesizing a variant nucleic acid library, wherein step ((1) comprises
`
`collecting results for at least 90% of the predetermined sequences. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein step (d) comprises collecting results
`
`for at least 100% of the predetermined sequences. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein at least about 70% of a predicted diversity is
`
`represented. Further provided herein are methods of synthesizing a variant nucleic acid library,
`
`wherein at least about 90% of a predicted diversity is represented. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein at least about 95% of a predicted
`
`diversity is represented. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library, wherein at least 80% of the at least 500 polynucleotide sequences are a correct size.
`
`Further provided herein are methods of synthesizing a variant nucleic acid library, wherein at least
`
`2
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`about 80% of the at least 500 polynucleotide sequences are each present in the variant nucleic acid
`
`library in an amount within 2X of a mean frequency for each of the polynucleotide sequences in the
`
`library. Further provided herein are methods of synthesizing a variant nucleic acid library further
`
`comprising collecting results from the assay in step (c) for predetermined sequences associated
`
`with an enhanced or reduced activity. Further provided herein are methods of synthesizing a
`
`variant nucleic acid library, wherein the activity is cellular activity. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein the cellular activity comprises
`
`reproduction, growth, adhesion, death, migration, energy production, oxygen utilization, metabolic
`
`activity, cell signaling, response to free radical damage, or any combination thereof. Further
`
`provided herein are methods of synthesizing a variant nucleic acid library, wherein the variant
`
`nucleic acid library encodes sequences for variant genes or fragments thereof. Further provided
`
`herein are methods of synthesizing a variant nucleic acid library, wherein the variant nucleic acid
`
`library encodes for at least a portion of an antibody, an enzyme, or a peptide. Further provided
`
`herein are methods of synthesizing a variant nucleic acid library, wherein the nucleic acid library
`
`encodes a guide RNA (gRNA). Further provided herein are methods of synthesizing a variant
`
`nucleic acid library, wherein the nucleic acid library encodes a siRNA, a shRNA, a RNAi, or a
`
`miRNA.
`
`[0008]
`
`Provided herein are methods for generating a combinatorial library of nucleic acids, the
`
`method comprising: (a) designing predetermined sequences encoding for: (i) a first plurality of
`
`polynucleotides, wherein each polynucleotide of the first plurality of polynucleotides encodes for
`
`variant sequence compared to a single reference sequence and (ii) a second plurality of
`
`polynucleotides, wherein each polynucleotide of the second plurality of polynucleotides encodes
`
`for variant sequence compared to the single reference sequence; (b) synthesizing the first plurality
`
`of polynucleotides and the second plurality of polynucleotides, and (c) mixing the first plurality of
`
`polynucleotides and the second plurality of polynucleotides to form the combinatorial library of
`
`nucleic acids, wherein at least about 70% of a predicted diversity is represented. Further provided
`
`herein are methods for generating a combinatorial library of nucleic acids, wherein the
`
`combinatorial library is a non-saturating combinatorial library. Further provided herein are
`
`methods for generating a combinatorial library of nucleic acids, wherein the combinatorial library
`
`is a saturating combinatorial library. Further provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein at least 10,000 polynucleotides are synthesized.
`
`Further provided herein are methods for generating a combinatorial library of nucleic acids,
`
`wherein a total number of polynucleotides for generation of the non-saturating combinatorial
`
`library is at least 25% less than the total number polynucleotides for generation of a saturating
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`combinatorial library. Further provided herein are methods for generating a combinatorial library
`
`of nucleic acids, wherein at least 80% of variants are a correct size. Further provided herein are
`
`methods for generating a combinatorial library of nucleic acids, wherein at least about 90% of a
`
`predicted diversity is represented. Further provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein at least about 95% of a predicted diversity is
`
`represented. Further provided herein are methods for generating a combinatorial library of nucleic
`
`acids, wherein the combinatorial library encodes for a first reference sequence or a second
`
`reference sequence. Further provided herein are methods for generating a combinatorial library of
`
`nucleic acids, wherein the combinatorial library when translated encodes for a protein library.
`
`Further provided herein are methods for generating a combinatorial library of nucleic acids,
`
`wherein the nucleic acids of the combinatorial library are inserted into vectors. Further provided
`
`herein are methods for generating a combinatorial library of nucleic acids further comprising
`
`performing PCR mutagenesis of a nucleic acid using the combinatorial library as primers for a PCR
`
`mutagenesis reaction. Further provided herein are methods for generating a combinatorial library
`
`of nucleic acids, wherein the combinatorial library encodes sequences for variant genes or
`
`fragments thereof. Further provided herein are methods for generating a combinatorial library of
`
`nucleic acids, wherein the combinatorial library encodes for at least a portion of an antibody, an
`
`enzyme, or a peptide. Further provided herein are methods for generating a combinatorial library
`
`of nucleic acids, wherein the combinatorial library encodes for at least a portion of a variable region
`
`or a constant region of the antibody. Further provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein the combinatorial library encodes for at least one
`
`CDR region of the antibody. Further provided herein are methods for generating a combinatorial
`
`library of nucleic acids, wherein the combinatorial encodes for a CDRl, a CDRZ, and a CDR3 on a
`
`heavy chain and a CDRl, a CDRZ, and a CDR3 on a light chain of the antibody. Further provided
`
`herein are methods for generating a combinatorial library of nucleic acids, wherein the
`
`combinatorial library encodes for a guide RNA (gRNA).
`
`[0009]
`
`Provided herein are methods of synthesizing a variant nucleic acid library, comprising:
`
`(a) providing predetermined sequences encoding for a plurality of polynucleotides, wherein the
`
`polynucleotides encode for a plurality of codons having a variant sequence compared to a single
`
`reference sequence; (b) selecting a distribution value for codons at a preselected position in the
`
`predetermined nucleic acid reference sequence; (c) providing machine instructions to randomly
`
`generate a set of nucleic acid sequences with a distribution value that aligns with the selected
`
`distribution value, wherein the set of nucleic acid sequences is less than the amount of nucleic acid
`
`sequences required to generate a saturating codon variant library; and (d) synthesizing the variant
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`nucleic acid library with a preselected distribution, wherein at least about 70% of a predicted
`
`diversity is represented. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library, wherein at least 80% of variants are a correct size. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein at least about 90% of a predicted diversity is
`
`represented. Further provided herein are methods of synthesizing a variant nucleic acid library,
`
`wherein at least about 95% of a predicted diversity is represented. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein the variant nucleic acid library
`
`when translated encodes for a protein library. Further provided herein are methods of synthesizing
`
`a variant nucleic acid library, wherein the nucleic acids of the variant nucleic acid library are
`
`inserted into vectors. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library further comprising performing PCR mutagenesis of a nucleic acid using the variant nucleic
`
`acid library as primers for a PCR mutagenesis reaction. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein a codon assignment is used for determining
`
`each codon of the plurality of codons having a variant sequence. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein the codon assignment is based on
`
`frequency of the codon sequence in an organism. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the organism is at least one of an animal, a
`
`plant, a fungus, a protist, an archaeon, and a bacterium. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the codon assignment is based on a diversity of
`
`the codon sequence.
`
`[0010]
`
`Provided herein are methods of synthesizing a variant nucleic acid library, comprising:
`
`(a) providing predetermined sequences encoding for a plurality of polynucleotides, wherein the
`
`polynucleotides encode for a codon having a variant sequence compared to a single reference
`
`sequence; (b) dividing the plurality of polynucleotides into 5’ fragments of polynucleotides and 3’
`
`fragments of polynucleotides, (c) selecting a distribution value for a codon at a preselected position
`
`in the predetermined nucleic acid reference sequence, (d) providing machine instructions to
`
`randomly generate a set of nucleic acids with a distribution value that aligns with the selected
`
`distribution value, wherein the set of nucleic acids is less than the amount of nucleic acids required
`
`to generate a saturating nucleic acid library; (6) synthesizing the 5’ fragments of polynucleotides
`
`and the 3’ fragments of polynucleotides; and (f) mixing the 5’ fragments of polynucleotides and the
`
`3 ’ fragments of polynucleotides to form the variant nucleic acid library, wherein at least about 70%
`
`of a predicted diversity is represented. Further provided herein are methods of synthesizing a
`
`variant nucleic acid library, wherein at least l0,000 polynucleotides are synthesized. Further
`
`provided herein are methods of synthesizing a variant nucleic acid library, wherein at least 80% of
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`variants are a correct size. Further provided herein are methods of synthesizing a variant nucleic
`
`acid library, wherein at least about 90% of a predicted diversity is represented. Further provided
`
`herein are methods of synthesizing a variant nucleic acid library, wherein at least about 95% of a
`
`predicted diversity is represented. Further provided herein are methods of synthesizing a variant
`
`nucleic acid library, wherein the plurality of polynucleotides is divided into at least one of more
`
`than one 5’ fragments and more than one 3’ fragments. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the variant nucleic acid library when translated
`
`encodes for a protein library. Further provided herein are methods of synthesizing a variant nucleic
`
`acid library, wherein the nucleic acids of the variant nucleic acid library are inserted into vectors.
`
`Further provided herein are methods of synthesizing a variant nucleic acid library further
`
`comprising performing PCR mutagenesis of a nucleic acid using the variant nucleic acid library as
`
`primers for a PCR mutagenesis reaction. Further provided herein are methods of synthesizing a
`
`variant nucleic acid library further comprising identifying a variant sequence with an enhanced or
`
`reduced activity. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library, wherein the activity is cellular activity. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the cellular activity comprises reproduction,
`
`growth, adhesion, death, migration, energy production, oxygen utilization, metabolic activity, cell
`
`signaling, response to free radical damage, or any combination thereof. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein the variant nucleic acid library
`
`encodes sequences for variant genes or fragments thereof. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the variant nucleic acid library encodes for at
`
`least a portion of an antibody, an enzyme, or a peptide. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the variant nucleic acid library encodes for at
`
`least a portion of a variable region or a constant region of the antibody. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein the variant nucleic acid library
`
`encodes for at least one CDR region of the antibody. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the variant nucleic acid library encodes for a
`
`CDRl, a CDR2, and a CDR3 on a heavy chain and a CDRl, a CDR2, and a CDR3 on a light chain
`
`of the antibody. Further provided herein are methods of synthesizing a variant nucleic acid library,
`
`wherein a number of different sequences synthesized in the variant nucleic acid library is in a range
`
`of 50 to 1,000,000. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library, wherein a number of different sequences synthesized in the variant nucleic acid library is in
`
`a range of 500 to 25000. Further provided herein are methods of synthesizing a variant nucleic acid
`
`library, wherein a number of different sequences synthesized in the variant nucleic acid library is in
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`a range of 1000 to 15000. Further provided herein are methods of synthesizing a variant nucleic
`
`acid library further comprising performing PCR mutagenesis of a nucleic acid using the variant
`
`nucleic acid library as primers for a PCR mutagenesis reaction. Further provided herein are
`
`methods of synthesizing a variant nucleic acid library, wherein a codon assignment is used for
`
`determining the codon having a variant sequence. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein the codon assignment is based on frequency of
`
`the codon sequence in an organism. Further provided herein are methods of synthesizing a variant
`
`nucleic acid library, wherein the organism is at least one of an animal, a plant, a fungus, a proti st,
`
`an archaeon, and a bacterium. Further provided herein are methods of synthesizing a variant
`
`nucleic acid library, wherein the codon assignment is based on a diversity of the codon sequence.
`
`Further provided herein are methods of synthesizing a variant nucleic acid library, wherein the
`
`nucleic acid library encodes a guide RNA (gRNA).
`
`[0011]
`
`Provided herein are methods for generating a combinatorial library of nucleic acids, the
`
`method comprising: (a) providing predetermined sequences encoding for: (i) a first plurality of
`
`polynucleotides, wherein each polynucleotide of the first plurality of polynucleotides encodes for a
`
`variant sequence compared to a single reference sequence and (ii) a second plurality of
`
`polynucleotides, wherein each polynucleotide of the second plurality of polynucleotides encodes
`
`for a variant sequence compared to the single reference sequence, (b) providing a structure having a
`
`surface, (c) synthesizing the first plurality of polynucleotides, wherein each polynucleotide of the
`
`first plurality of polynucleotides extends from the surface; (d) synthesizing the second plurality of
`
`polynucleotides, wherein each polynucleotide of the second plurality of polynucleotides extends
`
`from the surface; (e) releasing the first plurality of polynucleotides and the second plurality of
`
`polynucleotides from the surface; and (f) mixing the first plurality of polynucleotides and the
`
`second plurality of polynucleotides to form the combinatorial library of nucleic acids, wherein at
`
`least about 70% of a predicted diversity is represented. Further provided herein are methods for
`
`generating a combinatorial library of nucleic acids, wherein at least about 90% of a predicted
`
`diversity is represented. Further provided herein are methods for generating a combinatorial library
`
`of nucleic acids, wherein at least about 95% of a predicted diversity is represented.
`
`[0012]
`
`Provided herein are methods of synthesizing a variant nucleic acid library, comprising:
`
`(a) designing predetermined sequences encoding for a plurality of polynucleotides, wherein the
`
`polynucleotides encode for a plurality of codons having a variant sequence compared to a single
`
`reference sequence; (b) synthesizing the plurality of polynucleotides to generate the variant nucleic
`
`acid library, wherein at least about 70% of a predicted diversity is represented, (c) expressing the
`
`variant nucleic acid library; and (d) evaluating an activity associated with variant nucleic acid
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`library. Further provided herein are methods of synthesizing a variant nucleic acid library, wherein
`
`at least about 90% of a predicted diversity is represented. Further provided herein are methods of
`
`synthesizing a variant nucleic acid library, wherein at least about 95% of a predicted diversity is
`
`represented.
`
`[0013]
`
`Provided herein are methods for generating a combinatorial library of nucleic acids, the
`
`method comprising: (a) providing predetermined sequences encoding for: (i) a first plurality of
`
`non-identical polynucleotides, wherein each non-identical polynucleotide of the first plurality of
`
`non-identical pol ynucleotides encodes for a variant sequence compared to a single reference
`
`sequence and (ii) a second plurality of non-identical polynucleotides, wherein each non-identical
`
`polynucleotide of the second plurality of non-identical polynucleotides encodes for a variant
`
`sequence compared to the single reference sequence; (b) providing a structure having a surface; (c)
`
`synthesizing the first plurality of non-identical polynucleotides, wherein each non-identical
`
`polynucleotide of the first plurality of non—identical polynucleotides extends from the surface; (d)
`
`synthesizing the second plurality of non—identical polynucleotides, wherein each non—identical
`
`polynucleotide of the second plurality of non-identical polynucleotides extends from the surface;
`
`(6) releasing the first plurality ofnon-identical polynucleotides and the second plurality ofnon-
`
`identical polynucleotides from the surface; and (f) mixing the first plurality of polynucleotides and
`
`the second plurality of polynucleotides to form the combinatorial library of nucleic acids, wherein
`
`at least about 70% of a predicted diversity is represented. Provided herein are methods for
`
`generating a combinatorial library of nucleic acids, wherein the combinatorial library is a non-
`
`saturating combinatorial library. Provided herein are methods for generating a combinatorial
`
`library of nucleic acids, wherein the combinatorial library is a saturating combinatorial library.
`
`Provided herein are methods for generating a combinatorial library of nucleic acids, wherein at
`
`least 10,000 polynucleotides are synthesized. Provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein a total number of polynucleotides for generation of
`
`the non-saturating combinatorial library is at least 25% less than the total number polynucleotides
`
`for generation of a saturating combinatorial library. Provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein at least 80% of variants are a correct size. Provided
`
`herein are methods for generating a combinatorial library of nucleic acids, wherein the variant
`
`combinatorial library encodes for a first reference sequence or a second reference sequence.
`
`Provided herein are methods for generating a combinatorial library of nucleic acids, wherein the
`
`combinatorial library when translated encodes for a protein library. Provided herein are methods
`
`for generating a combinatorial library of nucleic acids, wherein the nucleic acids of the
`
`combinatorial library are inserted into vectors. Provided herein are methods for generating a
`
`

`

`WO 2018/170164
`
`PCT/U82018/022487
`
`combinatorial library of nucleic acids further comprising performing PCR mutagenesis of a nucleic
`
`acid using the combinatorial library as primers for a PCR mutagenesis reaction. Provided herein
`
`are methods for generating a combinatorial library of nucleic acids, wherein the combinatorial
`
`library encodes sequences for variant genes or fragments thereof. Provided herein are methods for
`
`generating a combinatorial library of nucleic acids, wherein the combinatorial library encodes for at
`
`least a portion of an antibody, enzyme, or peptide. Provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein the combinatorial library encodes for at least a
`
`portion ofa variable region or constant region of the antibody. Provided herein are methods for
`
`generating a combinatorial library of nucleic acids, wherein the combinatorial library encodes for at
`
`least one CDR region of the antibody. Provided herein are methods for generating a combinatorial
`
`library of nucleic acids, wherein the combinatorial encodes for a CDRl, CDR2, and CDR3 on a
`
`heavy chain and CDRl, CDR2, and CDR3 on a light chain of the antibody. Provided herein are
`
`methods for generating a combinatorial library of nucleic acids, wherein the combinatorial library
`
`encodes for guide RNA (gRNA). Provided herein are methods for generating a combinatorial
`
`library of nucleic acids, wherein the combinatorial library has an aggregate error rate of less than 1
`
`in 1000 bases compared to predetermined sequences. Provided herein are methods for generating a
`
`combinatorial library of nucleic acids, wherein the structure is a solid support, gel, or beads, and
`
`wherein the solid support is a plate or a column.
`
`[0014]
`
`Provided herein are methods of synthesizing a variant nucleic acid library, comprising:
`
`(a) providing predetermined sequences encoding for a plurality of non-identical polynucleotides,
`
`wherein the non—identical polynucleotides encode for a plurality of codons having a variant
`
`sequence compared to a single reference sequence; (b) selecting a distribution value for codons at a
`
`preselected position in the predetermined nucleic acid reference sequence; (c) providing machine
`
`instructions to randomly generate a set of nucleic acids, wherein the set of nucleic acids is less than
`
`the amount of nucleic acids required to generate a saturating codon variant library, and (d)
`
`synthesizing a nucleic acid library with a preselected distribution, wherein at least about 70% of a
`
`predicted diversity is represented. Provided herein are methods of synthesizing a variant