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/U82017/026232
`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 REPORT consists of a total of 9 sheets, including this cover sheet.
`
`In the attached sheets, any reference to the written opinion of the International Searching Authority should be read as a
`reference to the international preliminary report on patentability (Chapter I) instead.
`
`This report contains indications relating to the following items:
`
`Box \0. I
`
`Basis of the report
`
`Box \0. II
`
`Box \0.
`
`
`
`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 Colombettes
`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)
`
`AthHZEd officer
`
`Agnes WIttmann-Regls
`e—mail; pct.[eam6@wip0.int
`
`
`
`
`
`Box \0.
`
`Box \0.
`
`Box \0.
`
`Box \0.
`
`Box \0.
`
`E |
`
`:l
`
`X & g |
`
`:l
`
`|:l
`|:l
`
`

`

`PCT/USZO1 7I026232 28.08.201 7
`
`From the
`INTERNATIONAL SEARCHING AUTHORITY
`
`PATENT COOPERATION TREATY
`
`TO: David Harburger
`Wilson Sonsini Goodrich & Rosati
`650 Page Mill Road
`Palo Alto, California 94304
`United States of America
`
`PCT
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`(PCT Rule 43bis.l) Date ofmailing
`
`Applicant’s or agent’s file reference
`44854-727601
`
`(day/month/year) 2 8 A U G 201 7
`FOR FURTHER ACTION
`See paragraph 2 below
`
`International application No.
`
`International filing date (day/mamh/year)
`
`Priority date (day/monrhwear)
`
`22 August 2016 (22.08.2016)
`
`05 April 2017 (05.04.2017)
`PCT/US17/26232
`lntemational Patent Classification (IPC) or both national classification and IPC
`IPC - C12N15/10; 15/90 (2017.01)
`CPC -
`
`C12N 15/1082. 15/1079. 15/63, 15/1093, 15/907. 15/102, 9/22
`
`APP'iw“ TWIST BIOSCIENCE CORPORATION
`
`
`1. This opinion contains indications relating to the following items:
`
`EDDEEEDK
`
`Box No.
`
`I
`
`Basis ofthe opinion
`
`Box No. II
`
`Priority
`
`Box No. III
`
`Nonvestablishment ofopinion with regard to novelty, inventive step and industrial applicability
`
`Box No. IV
`
`Lack ofunity ofinvention
`
`Box No. V
`
`Box No. VI
`
`Reasoned statement under Rule 43bis. l(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`Certain documents cited
`
`Box No. Vll Certain defects in the intemational application
`
`Box No. VIII Certain observations on the international application
`
`2. FURTHER ACTION
`
`Ifa demand for international preliminary examination is made, this opinion will be considered to be a written opinion ofthe
`lntemational Preliminary Examining Authority (“IPEA”) except that this does not apply where the applicant chooses an Authority
`other than this one to be the IPEA and the chosen IPEA has notified the Intemational Bureau under Rule 66. lbis(b) that written
`opinions ofthis lntemational Searching Authority will not be so considered.
`Ifthis opinion is, as provided above, considered to be a written opinion ofthe IPEA, the applicant is invited to submit to the IPEA
`a written reply together, where appropriate, with amendments, before the expiration of} months from the date ofmailing of Form
`PCT/ISA/ZZO or before the expiration of22 months fi'om the priority date, whichever expires later.
`For further options, see Form PCT/lSA/ZZO.
`
`Name and mailing address ofthe ISA/US Date ofcompletion ofthis opinion
`Mail Stop PCT. Attn: ISA/US
`Commissioner for Patents
`P.O. Box 1450, Alexandria. Virginia 22313-1450
`Facsimile No. 571-273-8300
`
`09 AUQUSt 2017 (09082017)
`
`PCT 051:: 571-272-7774
`
`Authorized officer
`Shane Thomas
`PCT Helpdesk: 5712724300
`
`Form PCT/lSA/237 (cover sheet) (January 2015)
`
`

`

`PCT/USZO1 7I026232 28.08.201 7
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`lntemational application No.
`PCT/USl7/26232
`
`Box No. l
`
`Basis of this opinion
`
`1. With regard to the language, this opinion has been established on the basis of:
`
`El the international application in the language in which it was filed.
`E] a translation ofthe international application into
`furnished for the purposes ofintemational search (Rules 12.3(a) and 23.1(b)).
`
`which is the language ofa translation
`
`This opinion has been established taking into account the rectification ofan obvious mistake authorized by or notified to
`this Authority under Rule 9] (Rule 43bis. 1(a)).
`
`With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this opinion has
`been established on the basis ofa sequence listing:
`
`a,
`
`forming part ofthe international application as filed:
`
`E in the form ofan Annex C/ST.25 text file.
`:1 on paper or in the form ofan image file.
`
`5. Additional comments:
`
`b. E] furnished together with the intemational application under PCT Rule l3ter. l(a) for the purposes ofintemational
`search only in the form ofan Annex C/ST.25 text file.
`
`c. E] furnished subsequent to the international filing date for the purposes ofintemational search only:
`[:1 in the form of an Annex C/ST.25 text file (Rule l31eril(a)).
`D on paper or in the form ofan image file (Rule l3ter. l(b) and Administrative Instructions, Section 7l3).
`
`4. D In addition, in the case that more than one version or copy ofa sequence listing has been filed or furnished, the required
`statements that the information in the subsequent or additional copies is identical to that forming part ofthe application as
`filed or does not go beyond the application as filed, as appropriate, were fumished.
`
`Form PCT/ISA/237 (Box No. l) (January 2015)
`
`

`

`PCT/USZO1 7/026232 28.08.201 7
`
`WRITTEN OPINION 0]: THE
`
`lntemational application No.
`
`INTERNATIONAL SEARCHING AUTHORITY
`
`PCT/US17/26232
`
`
`
`Box No. III 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), or to be industrially
`applicable have not been examined in respect of:
`
`E]
`
`the entire international application.
`
`
`)2 claims Nos. 10-20
`
`See Supplemental Box for further details.
`
`because:
`
`CI
`
`the said international application, or the said claims Nos.
`subject matter which does not require an international search (speci'fil):
`
`relate to the following
`
`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 (specifii):
`
`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).
`
`[j the claims, or said claims Nos.
`by the description that no meaningful opinion could be formed (specifiz):
`
`are so inadequately supported
`
`fl
`
`no international search report has been established for said claims Nos. 10: 20
`
`D a meaningful opinion could not be formed without the sequence listing; the applicant did not, within the prescribed time limit:
`furnish a sequence listing in the form of an Annex C/ST.25 text file, and such listing was not available to the
`lntemational Searching Authority in the fomi and manner acceptable to it; or the sequence listing fumished did not
`comply with the standard provided for in Annex C ofthe Administrative Instructions.
`fumish a sequence listing on paper or in the form ofan image file complying with the standard provided for in Annex
`C ofthe Administrative Instructions, and such listing was not available to the lntemational Searching Authority in the
`form and manner acceptable to it; or the sequence listing fumished did not comply with the standard provided for in
`Annex C ofthe Administrative Instructions.
`
`pay the required late furnishing fee for the furnishing of a sequence listing in response to an invitation under
`Rule l3ter. l(a) or (b).
`
`E]
`
`Fomi PCT/ISA/237 (Box No. III) (January 20l5)
`
`

`

`PCT/USZO1 7/026232 28.08.201 7
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/USt7/26232
`
`
`
`Box No. IV Lack of unity ofinvention
`
`l. '2 In response to the invitation (Form PCT/lSA/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.
`
`not paid additional fees.
`
`
`
`2. E] This Authority found that the requirement of unity ofinvention is not complied with and chose not to invite the applicant to
`pay additional fees.
`
`3. This Authority considers that the reguirement ofunity ofinvention in accordance with Rule 13.], 13.2 and 13.3 is
`
`D complied with.
`
`'2'
`
`not complied with for the following reasons:
`
`This application contains the following inventions or groups of inventions which are not so linked as to form a single general inventive
`concept under PCT Rule 13.1. In order for all inventions to be examined. the appropriate additional examination fees must be paid.
`
`Group I, Claims 1-9. 11-19, 21—26 and 47-62 are directed toward nucleic acid and amplicon libraries and methods for the synthesis
`thereof.
`
`Group II, Claims 27-46 are directed toward cell libraries.
`
`The inventions listed as Groups | and N 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 I include wherein at least about 80% of the 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. not present in Group II; the special technical
`features of Group II include depletion in expression of a gene, not present in Group I.
`
`Groups I and It 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 Regents of the University of California
`(hereinafter 'California').
`
`California discloses DNA molecules encoding different gRNA sequences (a library of structurally 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 one of the inventions, and since all 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 ofthe following parts ofthe international application:
`
`[:1 all parts.
`
`& the parts relating to claims Nos. 1'9' 11'19' 21-26’ 47—62
`
`Form PCT/lSA/237 (Box No. lV) (January 2015)
`
`

`

`PCT/USZO1 7I026232 28.08.201 7
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`Intemational application No.
`PCTIUS17/26232
`
`Box No. V
`
`Reasoned statement under Rule 43bis.l(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`
`-"‘-Continued Within the Next Supplemental Box—""-
`
`Statement
`
`Novelty (N)
`
`Inventive step as)
`
`1-4.5]1-3.6-9,11-17,18/14-16,19,21-26,47—62
`NONE
`
`
`1-4, we, 6-9, 11-17, 18/14-16. 19, 21-26, 55-62
`47—54
`
`Industrial applicability (IA)
`
`1—4, 511-3, 6-9, 11-17, 18/14—16. 19, 21—26, 47-62
`NONE
`
`Citations and explanations:
`
`Claims 47-54 lack an inventive step under PCT Article 33(3) as being obvious over WO 2016/011080 A2 to The Regents of the University
`of California (hereinafter ‘California‘) in view of US 2016/0102322 A1 to Life Technologies Corporation, at al. (hereinafter ‘Life
`Technologies').
`
`Regarding claim 47, California discloses a method for synthesis of a gRNA library (generating a Ientiviral ngNA library; abstract;
`paragraph [0005]), comprising: (a) providing predetermined sequences for at least 500 non-identical DNA molecules (library of DNA
`sequences, SEQ ID NOS: 2205.305; paragraph [0007]), wherein each non-identical DNA molecule encodes for a gRNA (DNA
`sequences encode ngNA; paragraph [0007]); (b) synthesizing the at least 500 non»identica| 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 generate a library of gRNAs (generating an ngNA from expression of DNA polynucleotides; abstract; paragraphs [0097].
`[0100]). California also discloses a library of gRNA molecules with low numbers of errors (library of ngNAs with less than 1% errors;
`paragraph [0101]). but California does not disclose 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. Life Technologies discloses methods for
`producing gRNA wherein at least about 75% of the gRNAs in the library of gRNAs are error free compared to the DNA encoding them
`(reduction of gRNA error rate to zero compared to the error rate in the DNA template; paragraph [0329]).
`It would have been obvious to
`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 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, because the ability to reduce the error rate to near zero in a population of gRNA compared to their DNA templates as
`disclosed by Life Technologies would have allowed the gRNA library as generated from a DNA library as previously disclosed by
`California to attain an error rate 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.
`
`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 DNA into host cells; paragraphs [0071], [0097]) the at least ‘500 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 ngNA in a vector for 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 ngNAs having less than 1% error rate; paragraph [0101]) encoded by the at least 500
`non-identical DNA molecules (library of DNA sequences, SEQ ID N05: 2-205,305; paragraph [0007]) are present in the library of gRNAs
`(generation of high fidelity libraries; paragraphs [0086], [0101], [0195]).
`
`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 numbers of errors (library of ngNAs with less than 1% errors; paragraph [0101]) generated from
`predetermined sequences for the at least 500 non»identica| DNA molecules (library of DNA sequences. SEQ ID NOs: 2205.305;
`paragraph [0007]). but California does not disclose wherein at least 87% of the gRNAs in the library of gRNAs are error free compared to
`the predetermined sequences for the at least 500 non—identical DNA molecules. Life Technologies discloses methods for producing
`gRNA wherein at least about 87% of the gRNAs in the library of gRNAs are error free compared to the DNA encoding them (reduction of
`gRNA error rate to zero compared to the error rate in the DNA template; paragraph [0329]).
`it would have been obvious to 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
`gRNAs in the library of gRNAs are error free compared to the predetermined sequences for the at least 500 non-identical DNA
`molecules, because the ability to reduce the error rate to near zero in a population of gRNA compared to their DNA templates as
`disclosed by Life Technologies would have allowed the gRNA library as generated from a DNA library as previously disclosed by
`California to attain an error rate wherein at least about 87% of the gRNAs in the library of gRNAs are error free compared to the
`predetermined sequences for the at least 500 non—identical DNA molecules.
`
`Form PCT/lSA/237 (Box No. V) (January 20l5)
`
`

`

`PCT/USZO1 7I026232 28.08.201 7
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
` International application No.
`
`PCTIUS17/26232
`
`Supplemental Box
`
`
`
`in case the space in any of the preceding boxes is 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 DNA into host cells; paragraphs [0071], [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 DNA into 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 DNA into host cells; paragraphs [0071], [0097]) the at least 500 non-identical DNA
`
`molecules (library of DNA sequences, SEQ lD NOS: 2-205.305; paragraph [0007]) into cells (DNA transfected into HEK293 cells;
`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 Organism is Arabldopsls thaliana, Caenorhabditis elegans, Canis lupus familiaris, chlamydomonas reinhardtii, Dania rerio,
`
`
`
`Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli (insertion into E. coli cloning vector; paragraph [0099]), Homo
`
`sapiens (human target loci; paragraph [0012]), Alacaca mulatta, Mus musculus (mouse target loci; paragraph [0012]), Oryctolagus
`
`
`cuniculus, Rattus norvegicus, Saccharomyces cerevisiae. 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]) encodes for a single
`
`
`gRNA (DNA sequences encode ngNA; 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 meet the criteria set out in PCT Article 33(2)-(3).
`
`Claim 1 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 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.
`California discloses a nucleic acid library, 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
`sequence array; abstract; paragraph [0100]; Figure 1) encodes for a different gRNA sequence (DNA sequences encode ngNA;
`paragraph [0007]). California also discloses determining the frequency of each ngNA (paragraphs [0005], [0129]), but California does
`not disclose 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.
`
`
`
`
`
`
`
`
`
`Life Technologies discloses gRNA encoded by DNA templates (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% 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.
`
`
`
`WO 2015/040075 A1 to Genome Research Limited (hereinafter 'Genome Research') discloses constructing a mouse Ientiviral 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 gRNA in species in the library (page 8. lines 1-4; Figure 22), but Genome Research does not
`disclose 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.
` US 5,830,662 A to Scares et al. (hereinafter ‘Soares') discloses cDNA frequencies (1 :1:1 compared with other classes; column 1, lines
`
`
`54-62) and mRNA libraries having average (mean) frequencies calculated for each member of the classes of nucleic acid (column 40,
`
`lines 8-11; lines 20-21). Scares does not disclose 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.
`
`Therefore, it would not have been obvious to 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 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. because the disclosure of
`Soares indicating that the relative frequency of individual members or classes of a library of RNA species derived from a DNA template
`
`
`may be calculated neither teaches nor fairly suggests a determination of a level wherein at least about 80% of the at least 500
`non-identical DNA molecules are each present in the nucleic acid library, as previously disclosed by California, nor a determination ofan
`
`amount within 2x of a mean frequency for each of the non-identical DNA molecules in the library.
`Claims 2-4, 5/1-3, 6-9 and 11-13 also meet the criteria because of their dependence on positive claim 1.
`
`-"'-Continued Within the Next Supplemental Box-“‘-
`
`
`
`
`Form PCT/lSA/237 (Supplemental Box) (January 2015)
`
`

`

`PCT/USZO1 7I026232 28.08.201 7
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`
`International application No.
`PCT/US17/26232
`
`Supplemental Box
`
`In case the space in any ofthe preceding boxes is not sufficient.
`
`Continuation of:
` -“'"-Continued from Previous Supplemental Box-“"-
`
`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 ngNA sequence, wherein each ngNA 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.
`
` 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
`sequence array; abstract; paragraph [0100]; Figure 1) encodes for a different ngNA sequence (DNA sequences encode ngNA:
`paragraph [0007]), wherein each ngNA sequence comprises a targeting domain complementary to a eukaryotic gene (ngNA targeted
`
`
`to human or mouse loci; paragraph [0012]). California also discloses determining the frequency of each ngNA (paragraphs [0005],
`[0129]). but California 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 frequency for each of the non-identical nucleic acids in the library
`
`
`
`
`
`
` Genome Research discloses constructing a mouse Ientiviral 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 gRNA in species in the library
`
`
`(page 8. lines 1-4; Figure 22), but Genome Research 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 frequency for each of the non-identical
`
`nucleic acids in the library.
`
`
`
`
`
`
`
`
`Life Technologies discloses gRNA encoded by DNA templates (paragraph [0025]) and libraries of said RNA species (libraries of
`CRISPR system components; paragraph [0168]). Lll'e Technologies 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 frequency for each of the
`non-identical nucleic acids in the library.
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`Soares discloses cDNA frequencies (121:1 compared with other classes; column 1. lines 54-82) and mRNA libraries having average
`(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 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 frequency for each of the non-identical nucleic acids in the library.
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`Therefore, it would not have been obvious to one of ordinary skill in the art at the time of the invention to modify the disclosure of
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`California to provide wherein at least about 80% of the at least 2000 non-identical nucleic acids are each present in 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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`Scares indicating that the relative frequency of individual members or classes of a library of RNA species derived from a DNA template
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`may be calculated neither teaches nor fairly 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 present in the nucleic acid library, as previously disclosed by California, nor a determination of an
`amount within 2x of a mean frequency for each of the non-identical nucleic acids in the library.
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`Claims 15-17. 18/14-16 and 21-23 also meet the criteria because of their dependence on positive claim 14.
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` Claim 24 meets the criteria set out in PCT Article 33(2)—(3) because the prior art does not teach or fairly suggest an amplicon library.
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`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
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`mean frequency for each of the non—identical DNA molecules in the library.
` California discloses an amplicon library (libraries amplified by PCR; paragraph [0228]). wherein the amplicon library comprises a
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`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
`non-identical DNA molecule encodes for a different gRNA sequence (DNA sequences encode ngNA; paragraph [0007]). California
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`also discloses determining the frequency of each ngNA (paragraphs [0005], [0129]), but California does not disclose 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.
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` Life Technologies discloses gRNA encoded by DNA templates (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% of the plurality of non—identical DNA molecules are each present in the
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`amplicon library in an amount within 2x of a mean frequency for each of the non-identical DNA molecules in the library.
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` Genome Research discloses constructing a mouse Ientiviral gRNA library (page 51, lines 35-38) derived from DNA sequences encoding
`the gRNAs (abstract; page 4, lines 8-13) and amplicons derived from members of the library (page 31, lines 7-8). Genome Research
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`also discloses calculating frequencies of gRNA in species in 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 present in the amplicon library in an
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`amount within 2x of a mean frequency for each of the non-identical DNA molecules in the library.
` -“‘-Continued Within the Next Supplemental Box-"'-
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`Form PCT/lSA/237 (Supplemental Box) (January 2015)
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`Claims 55 meets the criteria set out in PCT Article 33(2)-(3) because the prior art does not teach or fairly suggest a method for synthesis
`of a gRNA library, comprising: (a) providing predetermined sequences for a plurality of non-identical DNA molecules, wherein each
`non-identical DNA molecule encodes for a gRNA; (b) providing a surface, wherein the surface comprises clusters of loci for nucleic acid
`extension reaction; (c) synthesizing the plurality of non-identical DNA molecules, wherein each nonidentical DNA molecule extends from
`the surface; and (d) transferring the plurality of non-identical DNA molecules into cells.
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`PCT/USZO1 7I026232 28.08.201 7
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`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
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` lntemational application No.
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`PCT/USl7l26232
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`Supplemental Box
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`in case the space in any of the preceding boxes is not sufficient.
`Continuation of:
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` -“'-Continued from Previous Supplemental Box-“'-
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`Soares discloses cDNA frequencies (1 :1 :1 compared with other classes; column 1, lines 54-62) and mRNA libraries having average
`(mean) frequencies calculated for each member of the classes of nucleic acid (column 40, lines 8—1 1; lines 20-21). Scares does not
`disclose wherein at least about 80% of the plurality of non-identical DNA molecules are each present in the amplicon library in an
`amount withi