PATENT COOPERATION TREATY
`
`PCT
`
`INTERNATIONAL PRELIMINARY REPORT ON PATENTABILITY
`(Chapter I of the Patent Cooperation Treaty)
`
`(PCT Rule 44bis)
`
`Applicant’s or agent’s file reference
`44854-727601
`
`FOR FURTHER ACTION
`
`See item 4 below
`
`International filing date (day/month/year)
`International application No.
`05 April 2017 (05.04.2017)
`PCT/US2017/026232
`International Patent Classification (8th edition unless older edition indicated)
`See relevant information in Form PCT/ISA/237
`
`Priority date (day/month/year)
`22 August 2016 (22.08.2016)
`
`Applicant
`TWIST BIOSCIENCE CORPORATION
`
`This international preliminary report on patentability (Chapter I) is issued by the International Bureau on behalf of the
`International Searching Authority under Rule 44 bis.1(a).
`
`This REPORTconsists of a total of 9 sheets, including this cover sheet.
`
`In the attached sheets, any reference to the wrilten opinion of the International Searching Authority should be read as a
`reference to the international preliminary report on patentability (Chapter I) instead.
`
`Box No. I
`
`Box No. II
`
`
`
`Xx
`
`4 L
`
`I]
`LI
`LI
`
`
`
`This report contains indications relating to the following items:
`Xx
`CI
`Xx
`
`
`
`Box No. I
`
`Box No. IV
`
`Box No. V
`
`Box No. VI
`
`Box No.
`
`Box No.
`
`Basis of the report
`
`Priority
`
`Non-establishment of opinion with regard to novelty, inventive step and industrial
`applicability
`
`Lack of unity of invention
`
`Reasoned statement under Article 35(2) with regard to novelty, inventive step or
`industrial applicability; citations and explanations supporting such statement
`
`Certain documents cited
`
`Certain defects in the international application
`
`Certain observations on the international application
`
`‘Lhe International Bureau will communicate this report to designated Offices in accordance with Rules 44bis.3(c) and 93bis.1
`but not, except where the applicant makes an express request under Article 23(2), before the expiration of 30 months from
`the priority date (Rule 44bis .2).
`
`The International Bureau of WIPO
`34, chemin des Colombcttes
`1211 Geneva 20, Switzerland
`Facsimile No. +41 22 338 82 70
`Form PCT/IB/373 (January 2004)
`
`Date of issuance of this report
`26 February 2019 (26.02.2019)
`Authorized officer
`Agnes Wittmann-Regis
`e-mail: pct.team6@wipo.int
`
`

`

`PCT/US2017/026232 28.08.2017
`
`PATENT COOPERATION TREATY
`
`From the
`INTERNATIONAL SEARCHING AUTHORITY
`
`To: David Harburger
`
`Wilson Sonsini Goodrich & Rosati
`650 PageMill Road
`Palo Alto, California 94304
`United States of America
`
`
`
`Applicant’s or agent’s file reference
`44854-727601
`
`PCT
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`(PCT Rule 43dis.1)
`
`Date of mailing
`(day/month/year) 2 8 A U G 201 ia
`FOR FURTHER ACTION
`See paragraph 2 below
`
`
`
`International filing date (day/month/year)
`International application No.
`05 April 2017 (05.04.2017)
`PCT/US17/26232
`International Patent Classification (IPC) or both national classification and IPC
`IPC - C12N 15/10; 15/90 (2017.01)
`CPC -
`
`C12N 15/1082, 15/1079, 15/63, 15/1093, 15/907, 15/102, 9/22
`
`Priority date (day/month/year)
`22 August 2016 (22.08.2016)
`
`Applicant Twist BIOSCIENCE CORPORATION
`
`
`
`1. This opinion contains indications relating to the following items:
`
`Box No. I
`
`Basis of the opinion
`
`Box No. Il
`
`Priority
`
`Box No. IV__Lack ofunity of invention
`
`Box No. HI Non-establishment ofopinion with regard to novelty, inventive step and industrial applicability
`
`NOUWRKOR Box No. VIII Certain observations on the international application
`
`Box No. V
`
`Box No. VI
`
`Reasonedstatement under Rule 434/s. 1(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`Certain documents cited
`
`Box No. VII Certain defects in the international application
`
`2. FURTHER ACTION
`
`If a demand for international preliminary examination is made, this opinion will be considered to be a written opinion of the
`International Preliminary Examining Authority (“IPEA”) exceptthat this does not apply where the applicant chooses an Authority
`other than this one to be the IPEA and the chosen IPEA hasnotified the International Bureau under Rule 66.1/s(b) that written
`opinions of this International Searching Authority will not be so considered.
`If this opinionis, as provided above, considered to be a written opinion of the IPEA, the applicantis invited to submit to the IPEA
`a written reply together, where appropriate, with amendments, before the expiration of 3 months from the date of mailing of Form
`PCT/ISA/220 or before the expiration of 22 months from the priority date, whichever expireslater.
`For further options, see Form PCT/ISA/220.
`
`PCT OSP: 571-272-7774
`
`Nameand mailing address of the ISA/US| Date of completion ofthis opinion
`Mail Stop PCT, Attn: ISA/US
`Commissioner
`for
`Patent:
`.
`P.O.Box1450, Alexandria, Virginia 22313-1450
`Facsimile No. 571-273-8300
`
`09 August 2017 (09.08.2017)
`
`Authorized officer
`Shane Thomas
`PCTHelpdesk: $7 1-272-4300
`
`Form PCT/ISA/237 (cover sheet) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US17/26232
`
`Box No. 1
`
`Basis of this opinion
`
`1. With regard to the language, this opinion has been established on the basis of:
`[xX]
`the international application in the language in whichit was filed.
`[| a translation of the international application into
`furnished for the purposes of international search (Rules 12.3(a) and 23.1(b)).
`
`whichis the languageofa translation
`
`2. C] This opinion has been establishedtaking into accountthe rectification of an obvious mistake authorizedby ornotified to
`this Authority under Rule 91 (Rule 43 dis. 1(a)).
`
`3.
`
`[xX] With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this opinion has
`been established on the basis of a sequencelisting:
`
`a.
`
`forming part of the international application as filed:
`S|
`in the form of an Annex C/ST.25 textfile.
`C] on paperorin the form of an imagefile.
`b. [] furnished together with the international application under PCT Rule 13¢er.1(a) for the purposesofinternational
`search only in the form of an Annex C/ST.25 text file.
`c. | furnished subsequentto the internationalfiling date for the purposesof international search only:
`[] in the form of an Annex C/ST.25 text file (Rule 13zer.1(a)).
`CL) on paperorin the form of an imagefile (Rule 13¢er.1(b) and Administrative Instructions, Section 713).
`
`Additional comments:
`
`4. J In addition, in the case that more than one version or copy of a sequencelisting has beenfiled or furnished, the required
`statements that the information in the subsequentor additional copies is identical to that forming part of the application as
`filed or does not go beyond the application asfiled, as appropriate, were furnished.
`
`Form PCT/ISA/237 (Box No. 1) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US17/26232
`
`
`
`Box No. II} =Non-establishment of opinion with regard to novelty, inventive step and industrial applicability
`
`The questions whether the claimed invention appears to be novel, to involve an inventive step (to be non obvious), orto be industrially
`applicable have not been examinedin respectof.
`[j the entire international application.
`
`xX claims Nos. 19, 20
`
`because:
`
`the said international application, or the said claims Nos.
`subject matter which does not require an international search (specify):
`
`relate to the following
`
`[] See Supplemental Box for further details.
`
`[] a meaningful opinion could not be formed withoutthe sequence listing; the applicant did not, within the prescribedtimelimit:
`furnish a sequence listing in the form of an Annex C/ST.25 text file, and such listing was not available to the
`International Searching Authority in the form and manner acceptable to it; or the sequencelisting furnished did not
`comply with the standard provided for in Annex C of the Administrative Instructions.
`furnish a sequencelisting on paper or in the form of an imagefile complying with the standard provided for in Annex
`C of the Administrative Instructions, and suchlisting was notavailable to the International Searching Authority in the
`form and manneracceptable to it; or the sequencelisting furnished did not comply with the standard provided forin
`Annex C of the Administrative Instructions.
`
`~<|
`
`the description, claims or drawings (indicate particular elements below) or said claims Nos. 10, 20
`are so unclear that no meaningful opinion could be formed (specify):
`
`Claims Nos. 10 and 20 are dependent claims which are not drafted in accordance with the second and third sentences of Rule 6.4(a).
`
`the claims, or said claims Nos.
`by the description that no meaningful opinion could be formed (specify):
`
`are so inadequately supported
`
`PX}
`
`no international search report has been established for said claims Nos, 10, 20
`
`pay the required late furnishing fee for the furnishing of a sequence listing in response to an invitation under
`Rule 13¢er.1(a) or (b).
`
`Form PCT/ISA/237 (Box No. III) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US17/26232
`
`
`
`Box No. IV Lackof unity of invention
`
`1, x] in response to the invitation (Form PCT/ISA/206)to pay additional fees the applicant has, within the applicable time limit:
`paid additional fees.
`
`paid additional fees under protest and, where applicable, the protest fee.
`
`paid additional fees under protest but the applicable protest fee was not paid.
`
`3. This Authurity considers that the requirement of unity of invention in accordance with Rule 13.1, 13.2 and 13.3 is
`[J complied with.
`x] not complied with for the following reasons:
`This application contains the following inventions or groupsof inventions which are not so linked asto form a single general inventive
`concept under PCT Rule 13.1. In orderfor ali inventions to be examined, the appropriate additional examination fees must be paid.
`
`<1 the parts relating to claims Nos. 1-9, 11-19, 21-26, 47-62
`
`not paid additional fees.
`2.[] This Authority found that the requirement of unity of invention is not complied with and chose notto invite the applicantto
`pay additional fees.
`
`Group |, Claims 1-9, 11-19, 21-26 and 47-62 are directed toward nucleic acid and ampliconlibraries and methodsfor the synthesis
`thereof.
`
`Group Il, Claims 27-46 are directed toward cell libraries.
`
`Theinventions listed as Groups | andII do not relate to a single general inventive concept under PCT Rule 13.1 because, under PCT
`Rule 13.2, they lack the same or corresponding special technical features for the following reasons: the special technical features of
`Group| include wherein at least about 80% of the non-identical DNA molecules are each presentin the nucleic acid library in an amount
`within 2x of a mean frequency for each of the non-identical DNA moleculesin thelibrary, not present in Group II; the special technical
`features of GroupII include depletion in expression of a gene, not present in Group|.
`
`Groups| andI! share the technical features including: DNA molecules encoding different gRNA sequences.
`
`However, these shared technical features are previously disclosed by WO 2016/011080 A2 to The Regentsof the University of California
`(hereinafter 'California’).
`
`California discloses DNA molecules encoding different gRNA sequences(a library ofstructurally distinct expression cassette-encoded
`small guide RNAs (DNA molecules encoding different gRNA sequences); paragraphs [0005], [0017)).
`
`Since none of the special technical features of the Groups| and II inventions is found in more than oneofthe inventions, andsinceall of
`the shared technical features are previously disclosed by the California reference,unity of invention is lacking.
`
`Consequently, this opinion has been established in respect of the following parts of the international application:
`[] all parts.
`
`Form PCT/ISA/237 (Box No. IV) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US17/26232
`
`Box No. V
`
`Reasoned statement under Rule 436/s.1(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`
`Statement
`
`Novelty (N)
`
`Inventive step (IS)
`
`Claims
`Claims
`
`Claims
`Claims
`
`1-4, 5/1-3, 6-9, 41-17, 18/14-16, 19, 21-26, 47-62
`NONE
`
`
`1-4, 5/1-3, 6-9, 11-17, 18/14-16, 19, 21-26, 55-62
`47-54
`
`Industrial applicability (1A)
`
`Claims
`Claims
`
`1-4, 5/1-3, 6-9, 11-17, 18/14-16, 19, 21-26, 47-62
`NONE
`
`-"**.-Continued Within the Next Supplemental Box-***-
`
`Regarding claim 50, California and Life Technologies in combination disclose the method of claim 47, and California also discloses a
`library of gRNA molecules with low numbersof errors(library of sgRNAs with less than 1%errors; paragraph [0101]) generated from
`predetermined sequencesfor the at least 500 non-identical DNA molecules(library of DNA sequences, SEQ ID NOs: 2-205,305;
`paragraph [0007)), but California does not disclose wherein at least 87% of the gRNAsin the library of gRNAsareerror free compared to
`the predetermined sequencesfor the at least 500 non-identical DNA molecules. Life Technologies discloses methods for producing
`gRNAwhereinatleast about 87% of the gRNAsin the library of gRNAsare error free compared to the DNA encoding them (reduction of
`gRNAerrorrate to zero comparedto the error rate in the DNA template; paragraph (0329]).
`It would have been obviousto one of
`ordinary skill in the art at the time of the invention to modify the disclosure of California to provide wherein at least about 87% of the
`Q@RNAsinthe library of gRNAsare error free compared to the predetermined sequencesforthe at least 500 non-identical DNA
`molecules, becausethe ability to reduce the error rate to near zero in a population of gRNA comparedto their DNA templates as
`disclosed by Life Technologies would have allowed the gRNAlibrary as generated from a DNAlibrary as previously disclosed by
`California to attain an error rate wherein at least about 87% of the gRNAsin thelibrary of gRNAsare error free comparedto the
`predetermined sequencesfor the at least 500 non-identical DNA molecules.
`
`Citations and explanations:
`
`Claims 47-54 lack an inventive step under PCTArticle 33(3) as being obvious over WO 2016/011080 A2 to The Regentsof the University
`of California (hereinafter‘California’) in view of US 2016/0102322 A1 to Life Technologies Corporation, et al. (hereinafter‘Life
`Technologies’).
`
`Regarding claim 47, California discloses a method for synthesis of a gRNAlibrary (generating a lentiviral sgRNA library; abstract;
`paragraph [0005}), comprising: (a) providing predetermined sequencesfor at least 500 non-identical DNA molecules(tibrary of DNA
`sequences, SEQ ID NOs: 2-205,305; paragraph [0007]), wherein each non-identical DNA molecule encodes for a gRNA (DNA
`sequences encode sgRNA; paragraph [0007]); (b) synthesizing the at least 500 non-identical DNA molecules(structurally distinct DNA
`sequence array synthesis on a slide surface; abstract; paragraph [0100]; Figure 1) and (c) transcribing the at least 500 non-identical DNA
`molecules to generatealibrary of gRNAs (generating an sgRNA from expression of DNA polynucleotides; abstract; paragraphs [0097],
`[0100]). California also disclosesa library of gRNA molecules with low numbersof errors(library of sgRNAswith less than 1% errors;
`paragraph [0101]), but California does not disclose wherein at least about 75% of the gRNAsin the library of gRNAsare errorfree
`comparedto the predetermined sequences for the at least 500 non-identical DNA molecules. Life Technologies discloses methods for
`producing gRNA wherein at least about 75% of the gRNAsin thelibrary of gRNAsare error free compared to the DNA encoding them
`(reduction of gRNAerror rate to zero comparedto the error rate in the DNA template; paragraph [0329]).
`It would have been obviousto
`oneofordinary skill in the art at the time of the invention to modify the disclosure of California to provide wherein at least about 75% of
`the gRNAsin thelibrary of gRNAsare error free compared to the predetermined sequencesforthe at least 500 non-identical DNA
`molecules, becausethe ability to reduce the error rate to near zero in a population of gRNA comparedto their DNA templates as
`disclosed by Life Technologies would have allowed the gRNAlibrary as generated from a DNAlibrary as previously disclosed by
`California to attain an error rate wherein at least about 75% of the gRNAsin the library of gRNAsare error free comparedto the
`predetermined sequencesforthe at least 500 non-identical DNA molecules.
`
`Regarding claim 48, California and Life Technologies in combination disclose the method of claim 47, and California also discloses (the
`method) further comprising transferring (incorporating DNAinto host cells; paragraphs [0071], [0097]) the at least500 non-identical DNA
`molecules (library of DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007)) into cells (DNA transfected into HEK293 cells;
`paragraph [0229])prior to the transcribing step (polynucleotides encoding sgRNAin a vectorfor integration into a host cell (paragraph
`[0097)).
`
`Regarding claim 49, California and Life Technologies in combination disclose the method of claim 47, and California also discloses
`wherein at least 96% of the gRNAs(library of sgRNAs having less than 1%error rate; paragraph [0101]) encoded by the at least 500
`non-identical DNA molecules(library of DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007)) are presentin the library of gRNAS
`(generationof high fidelity libraries; paragraphs [0086], [0101), (0195)).
`
`Form PCT/ISA/237 (Box No. V) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
` International application No. WRITTEN OPINION OF THE
`
`PCT/US17/26232
`
`INTERNATIONAL SEARCHING AUTHORITY
`
`Supplemental Box
`
`
`
`
`
`In case the space in any of the preceding boxesis not sufficient.
`Continuation of:
`
`
`-***.Continued from Box V: Citations and Explanations-***-
`
`
`
` Regarding claim 51, California and Life Technologies in combination disclose the method of claim 47, and California also discloses (the
`method) further comprising inserting (incorporating DNAinto hostcells; paragraphs (007 1], [0097]) the at least 500 non-identical DNA
`
`
`molecules(library of DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007)) into vectors (insertion of DNAinto vectors;
`
`paragraphs [0097], {0099)).
`
`
`Regarding claim 52, California and Life Technologies in combination disclose the method of claim 47, and California also discloses (the
`method) further comprising transferring (incorporating DNAinto host cells; paragraphs [0071], [0097]) the at least 500 non-identical DNA
`
`molecules(library of DNA sequences, SEQ 1D NOs: 2-205,305; paragraph [0007)) into cells (DNA transfected into HEK293cells;
`
`
`paragraph [0229]}) of an organism (insertion into E. coli cloning vector; paragraph [0099)).
`
`Regarding claim 53, California and Life Technologies in combination disclose the method of claim 52, and California also discloses
`
`
`wherein the oryanisrn is Arabldopsis thaliana, Caenorhabditis elegans, Canis lupus familiaris, Chlamydomonasreinhardtii, Dania rerio,
`
`Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli (insertion into E. coli cloning vector; paragraph [0099]), Homo
`
`
`
`sapiens (humantargetloci; paragraph (0012]), Alacaca mulatta, Mus musculus (mousetarget loci; paragraph [0012]), Oryctolagus
`cuniculus, Rattus norvegicus, Saccharomycescerevisiae, or Sus scrofa.
`
`Regarding claim 54, California and Life Technologies in combination disclose the method of claim 47, and California also discloses
`
`
`wherein each non-identical DNA molecule (library of DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007}) encodesfor a single
`
`gRNA(ONA sequences encode sgRNA; paragraph [0007}) or a dual gRNA.
`
` Claims 1-4, 5/1-3, 6-9, 11-17, 18/14-16, 19, 21-26 and 55-62 meetthecriteria set out in PCT Article 33(2)-(3).
`
`
`
`Claim 1 meetsthecriteria set out in PCT Article 33(2)-(3) because the prior art does not teachorfairly suggest a nucleic acid library,
`wherein the nucleic acid library comprisesat least 500 non-identical DNA mofecules, 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 presentin the
`
`nucleic acid library in an amount within 2x of a mean frequencyfor each of the non-identical DNA moleculesin the library.
`
`
`California discloses a nucleic acidlibrary, wherein the nucleic acid library comprises at least 500 non-identical DNA molecules(library of
`DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007]), wherein each non-identical DNA molecule (structurally distinct DNA
`sequencearray; abstract; paragraph {0100}; Figure 1) encodesfor a different gRNA sequence (DNA sequences encode sgRNA;
`paragraph (0007}). California also discloses determining the frequency of each sgRNA (paragraphs [0005], [0129]), but California does
`
`
`not disclose wherein at least about 80% ofthe at least 500 non-identical DNA molecules are each presentin the nucleic acid library in an
`amountwithin 2x of a mean frequency for each of the non-identical DNA moleculesin the library.
`
`Life Technologies discloses gRNA encoded by DNAtemplates (paragraph [0025)) and libraries of said RNA species(libraries of
`CRISPR system components; paragraph [0168]). Life Technologies does not disclose wherein at least about 80% ofthe at least 500
`
`
`non-identical DNA molecules are each presentin the nucleic acid library in an amountwithin 2x of a mean frequency -for each of the
`
`
`non-identical DNA moleculesin thelibrary.
`
`
`
`WO 2015/040075 A1 to Genome Research Limited (hereinafter ‘Genome Research’) discloses constructing a mouselentiviral gRNA
`
`
`library (page 51, lines 35-38) derived from DNA sequences encoding the gRNAs(abstract; page 4, lines 8-13). Genome Research also
`
`discloses calculating frequencies of gRNAin speciesin thelibrary (page 8, lines 1-4; Figure 22), but Genome Research does not
`
`
`disclose wherein at least about 80% ofthe at least 500 non-identical DNA molecules are each presentin the nucleic acid library in an
`amountwithin 2x of a mean frequency for each of the non-identical DNA moleculesin the library.
`
`
` US 5,830,662 A to Soaresetal. (hereinafter ‘Soares’) discloses cDNA frequencies (1:1:1 compared with other classes; column 1, lines
`54-62) and mRNAlibraries having average (mean) frequencies calculated for each memberofthe classes of nucleic acid (column 40,
`
`lines 8-11; lines 20-21). Soares does not disclose wherein at least about 80% of the at least 500 non-identical DNA molecules are each
`
`presentin the nucleic acid tibrary in an amountwithin 2x of a mean frequency for each of the non-identical DNA moleculesin thelibrary.
`
`
`Therefore, it would not have been obviousto one of ordinary skill in the art at the time of the invention to modify the disclosure of
`California to provide wherein at least about 80% of the at least 500 non-identical DNA molecules are each presentin the nucleic acid
`
`library in an amountwithin 2x of a mean frequencyfor each of the non-identical DNA moleculesin the library, because the disclosure of
`
`
`Soaresindicating that the relative frequency of individual members or classesof a library of RNA species derived from a DNA template
`may be calculated neither teaches norfairly suggests a determination of a level wherein at least about 80% of the at least 500
`non-identical DNA molecules are each presentin the nucleic acid library, as previously disclosed by California, nor a determination of an
`
`amountwithin 2x of a mean frequency for each of the non-identical DNA moleculesin thelibrary.
`-***-Continued Within the Next Supplemental Box-***-
`
`
`
`
`
`
`
`
`
`Claims 2-4, 5/1-3, 6-9 and 11-13 also meetthe criteria becauseof their dependence onpositive claim 1.
`
`
`
`
`Form PCT/ISA/237 (Supplemental Box) (January 2015)
`
`

`

`PCT/US2017/026232 28.08.2017
`
` WRITTEN OPINION OF THE
`International application No.
`
`INTERNATIONAL SEARCHING AUTHORITY
`PCT/US17/26232
`
`Supplemental Box
`
`In case the space in any of the preceding boxesis not sufficient.
`Continuation of:
`
`Claim 14 meets the criteria set out in PCT Article 33(2)-(3) because the prior art does not teach or fairly suggest a nucleic acid library,
`wherein the nucleic acid library comprises at least 2000 non-identical nucleic acids, wherein each non-identical nucleic acid encodes for
`a different sgRNA sequence, wherein each sgRNA sequence comprises a targeting domain complementary to a eukaryotic gene, and
`wherein at least about 80% ofthe at least 2000 non-identical nucleic acids are presentin the nucleic acid library in an amountwithin 2x
`of a mean frequencyfor each of the non-identical nucleic acidsin the library.
`
`
`
`
`
`
`Life Technologies discloses gRNA encoded by DNA templates (paragraph [0025)) and libraries of said RNA specias(libraries of
`CRISPR system components; paragraph [0168]). Life Technologies does not disclose wherein at least about 80%ofthe at least 2000
`non-identical nucleic acids are each presentin the nucleic acid library in an amountwithin 2x of a mean frequencyfor eachof the
`non-identical nucleic acidsin the library.
`
` California discloses a nucleic acid library, wherein the nucleic acid library comprises at least 2000 non-identical nucleic acids (library of
`DNA sequences, SEQ ID NOs: 2-205,305; paragraph [0007]), wherein each non-identical nucleic acid (structurally distinct DNA
`sequencearray; abstract; paragraph (0100); Figure 1) encodesfor a different sgRNA sequence (DNA sequences encode sgRNA;
`paragraph [0007]), wherein each sgRNA sequence comprises a targeting domain complementary to a eukaryotic gene (sgRNAtargeted
`to human or mouseloci; paragraph [0012]). California also discloses determining the frequency of each sgRNA (paragraphs [0005],
`[0129]), but Catifornia does not disclose wherein at least about 80% of the at least 2000 non-identical nucleic acids are each present in
`the nucleic acid library in an amount within 2x of a mean frequencyfor each of the non-identical nucleic acidsin the library
`
`
`
` -***-Continued from Previous Supplemental Box-***-
`
`
`
`
`
`
`
`
`
`
`
`
`
` GenomeResearch discloses constructing a mouselentiviral gRNAlibrary (page 51, lines 35-38) derived from DNA sequences encoding
`the gRNAs(abstract; page 4, lines 8-13). Genome Research also discloses calculating frequencies of gRNAin species in the library
`
`
`(page8, lines 1-4; Figure 22), but Genome Research doesnot disclose wherein at least about 80%of the at least 2000 non-identicat
`
`
`nucleic acids are each presentin the nucleic acid library in an amountwithin 2x of a mean frequencyfor each of the non-identical
`nucleic acids in the library.
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`Soares discloses cDNAfrequencies (1:1:1 compared with other classes; column 1, lines 54-62) and MRNAlibraries having average
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`(mean) frequencies calculated for each member of the classes of nucleic acid (column 40, lines 8-11; lines 20-21). Soares does not
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`disclose wherein at feast about 80% ofthe at least 2000 non-identical nucleic acids are each presentin the nucleic acidlibrary in an
`amountwithin 2x of a mean frequency for each of the non-identical nucleic acidsin thelibrary.
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`Therefore, it would not have been obviousto oneofordinary skill in the art at the time of the invention to modify the disclosure of
`California to provide wherein at least about 80%of the at least 2000 non-identical nucleic acids are each presentin the nucleic acid
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`library in an amount within 2x of a mean frequency for each of the non-identical nucleic acids in the library, because the disclosure of
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`Soaresindicating that the relative frequency ofindividual members or classesofa library of RNA species derived from a DNA template
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`maybe calculated neither teaches norfairly suggests a determination of a level wherein at least about 80% of the at least 2000
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`non-identical nucleic acids are each presentin the nucleic acid library, as previously disclosed by California, nor a determination of an
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`amount within 2x of a mean frequency for each of the non-identical nucleic acidsin the library.
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`Claims 15-17, 18/14-16 and 21-23 also meetthe criteria because of their dependenceon positive claim 14.
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` Claim 24 meetsthe criteria set out in PCT Article 33(2)-(3) because the prior art does notteach orfairly suggest an amplicon library,
`wherein the ampliconlibraty cornprisesa plurality of non-identical DNA molecules, wherein each non-identical DNA is presentin a
`population of amplification products, wherein each non-identical DNA molecule encodesfor a different gRNA sequence, and wherein at
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`least about 80% ofthe plurality of non-identical DNA molecules are each present in the amplicon library in an amountwithin 2x of a
`mean frequency for each of the non-identical DNA moleculesin thelibrary.
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` California discloses an amplicon library (libraries amplified by PCR: paragraph [0228)), wherein the amplicon library comprises a
`plurality of non-identical DNA molecules (library of DNA sequences, SEQ ID NOs:2-205,305; paragraph (0007)), wherein each
`non-identical DNA is present in a population of amplification products (libraries amplified by PCR; paragraph (0228]), wherein each
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`non-identical DNA molecule encodesfor a different gRNA sequence (DNA sequences encode sgRNA; paragraph [0007]). California
`also discloses determining the frequency of each sgRNA (paragraphs [0005], [0129]), but California does not disclose wherein at least
`about 80% ofthe plurality of non-identical DNA molecules are each presentin the amplicon library in an amount within 2x of a mean
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`frequency for each of the non-identical DNA moleculesin thelibrary.
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` Life Technologies discloses gRNA encoded by DNAtemplates (paragraph [0025)]) and libraries of said RNA species(libraries of
`CRISPR system components; paragraph [0168]) and amplicons derived from members of the library (paragraph [0025]). Life
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`Technologies does not disclose wherein at least about 80% ofthe plurality of non-identical DNA molecules are each presentin the
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`amplicon library in an amountwithin 2x of a medn frequency for each of the non-identical DNA moleculesin thelibrary.
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` Genome Researchdiscloses constructing a mouselentiviral gRNAlibrary (page 51, lines 35-38) derived from DNA sequences encoding
`the gRNAs(abstract; page4, lines 8-13) and amplicons derived from membersof the library (page 31, lines 7-8). Genome Research
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`also discloses calculating frequencies of gRNAin speciesin the library (page 8, lines 1-4; Figure 22), but Genome Research does not
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`disclose wherein at least about 80% of the plurality of non-identical DNA molecules are each presentin the amplicon library in an
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`amountwithin 2x of a mean frequencyfor each of the non-identical DNA moleculesin the library.
` -***-Continued Within the Next Supplemental Box-***-
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`Form PCT/ISA/237 (Supplemental Box) (January 2015)
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`PCT/US2017/026232 28.08.2017
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` International application No. WRITTEN OPINION OF THE
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`PCT/US17/26232
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`INTERNATIONAL SEARCHING AUTHORITY
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`Therefore, it would not have been obvious to oneof ordinary skill in the art at the time of the invention to modify the disclosure of
`California to provide whereinat least about 80% ofthe plurality of non-identical DNA molecules are each presentin the ampliconlibrary
`in an amountwithin 2x of a mean frequency for each of the non-identical DNA moleculesin the library, because the disclosure of Soares
`indicating that the relative frequencyof individual membersor classesof a library of RNA species derived fram a DNA template may be
`calculated neither teachesnorfairly suggests a determination of a level wherein at least about 80% ofthe plurality of non-identical DNA
`molecules at least are each presentin the amplicon library, as previously disclosed by California, nor a determination of an amount
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`within 2x of a mean frequency for each of the non-identical nucleic acids in the Ilbrary.
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` Claims 25 and 26 also meetthecriteria because of their dependenceon positive claim 24.
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`Claims 55 meets the criteria set out in PCT Article 33(2)-(3) because the prior art does not teach orfairly suggest a method for synthesis
`of a gRNAlibrary, comprising: (a} providing predetermined sequencesfora plurality of non-identical DNA molecules, wherein each
`non-identical DNA molecule encodes for a gRNA;(b) providing a surface, wherein the surface co