(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`UNDAIAAAAA
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`(43) International Publication Date
`26 March 2015 (26.03.2015)
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`WIPO!|PCT
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`Zz
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`(10) International Publication Number
`WO 2015/040075 Al
`
`(GD
`
`International Patent Classification:
`C12N 15/09 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/EP20 14/069825
`
`(22)
`
`International Filing Date:
`17 September 2014 (17.09.2014)
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`(25)
`
`Filing Language:
`
`English
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`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM,GT,
`HN, HR, HU,ID,IL,IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NL 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.
`
`(26)
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`(30)
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`Publication Language:
`
`English
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`(84)
`
`Priority Data:
`1316558.4
`18 September 2013 (18.09.2013)
`1321257.6
`2 December 2013 (02.12.2013)
`
`GB
`GB
`
`(72)
`
`Inventors: YUSA, Kosuke; Genome Research Limited,
`215 Euston Road, London Greater London NWI 2BE
`(GB). LI, Ylong; Genome Research Limited, 215 Euston
`Road, London Greater London NW1 2BE (GB).
`
`(74)
`
`(81)
`
`Agents: SUTCLIFFE, Nicholas ect al.; Mewburn Ellis
`LLP, 33 Gutter Lane, London Greater London EC2V 8AS
`(GB).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available). AF, AG, AL, AM,
`
`with international search report (Art. 21(3))
`
`before the expiration of the lime limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`(54) Title: GENOMIC SCREENING METHODS USING RNA-GUIDED ENDONUCLEASES
`
`(57) Abstract: This invention relates to the genomic screening of libraries of mutant mammalian cells in which each cell has a target
`geneinactivated by expression of an RNA-guided endonuclease and a guide RNA molecule (gRNA)specific for the target gene. The
`library of mutant cells expresses gRNA molecules specific for a set of target genes and a target gene from theset of target genes is
`inactivated in each cell in the library. Mutant mammalian cells that display a test phenotype from said library are selected and one or
`more nucleic acid sequences that encode gRNA molecules identified in the selected cell population. From these nucleic acid se -
`quences, the target genes that mediate the test phenotype may be identified. Screening methods and librarics and vector populations
`for use in screening methods are provided.
`
`wo2015/040075A1|IITINNNATITINUITARCATTAAMAATA
`
`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), Furasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`(71)
`
`
`
`Applicant: RESEARCH~—LIMITEDGENOME DK, FE, ES, FI, FR, GB, GR, HR, HU,IF,IS, IT, LT, LU,
`[GB/GB]; 215 Euston Road, London Greater London NW1
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`2BE (GB).
`SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ,
`GW, KM,ML, MR,NE, SN, TD, TG).
`Published:
`
`
`
`
`
`
`
`

`

`WO 2015/040075
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`PCT/EP2014/069825
`
`
`Genomic Screening Methods using RNA-Guided Endonucleases
`
`Field
`
`
`This invention relates to functional genomic screening and,
`
`in
`
`particular, methods for genome wide loss-of-function screening in
`
`mammalian cells.
`
`Background
`
`
`Genomic screens have been useful
`
`and the identification of new targets for drug discovery.
`
`in the elucidation of gene function
`
`The diploid nature of the mammalian genome has hampered rapid and
`
`efficient production of mutant cells and organisms.
`
`stem cells (ESCs), homozygous mutants can be obtained by knocking out
`
`In mouse embryonic
`
`both alleles of the relevant gene with two rounds of gene targeting.
`
`However,
`
`this process takes at least 2 months and genome-wide mutant
`
`libraries cannot be easily generated by this method.
`
`RNAi libraries have been used to systematically target large sets of
`
`genes in the genome, allowing many genes to be interrogated
`
`
`
`
`
`simultaneously using high-throughput formats (Sims et al. Genome
`
`Biology 2011, 12:R104; Torns et al Nature Rev Drug Discov (2007)
`
`556-568, Campeau et al Briefings in Functional Genomics. 10 4 215-
`
`6
`
`226).
`
`
`
`However, RNAi libraries are limited by the efficacy of gene silencing.
`
`Since RNAi mediated suppression is rarely 100% efficient, not ali
`
`genes in an RNAi library are knocked down with sufficient efficiency
`
`the amount of
`
`to generate a detectable phenotype. Furthermore,
`
`suppression of different genes in RNAi libraries is not uniform,
`
`further limiting the effectiveness of RNAi libraries for functional
`
`screening and cells targeted by RNAi often display off target effects.
`
`Summary
`
`The present inventors have developed a knock-out approach using RNA-
`
`guided endonuclease technology that allows high-throughput genome-wide
`
`loss of function screening of both dominant and recessive genes in
`
`
`
`mamnalian cells.
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`10
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`15
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`20
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`30
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`35
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`

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`PCT/EP2014/069825
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`An aspect of the invention provides a method of genomic screening
`
`comprising;
`
`
`providing a library of mutant mammalian cells, each cell in the
`
`library expressing an RNA-guided endonuclease and a gRNA specific for
`
`a
`
`a target gene, such that
`
`the target gene is inactivated in the cell,
`
`wherein the Library expresses GRNA molecules specific for a set
`
`of target genes, such that a gene from the set of target genes is
`
`inactivated in each mutant mammalian cell in the library,
`
`selecting a population of cells from the library that display a
`
`10
`
`test phenotype,
`
`identifying one or more nucleic acid sequences encoding gRNA
`
`molecules in the selected cell population, and;
`
`
`
`identifying from the one or more identified nucleic acid
`
`sequences one or more target genes that are inactivated in the
`
`15
`
`selected cell population.
`
`The target genes that are identified are candidate modulators of the
`
`
`
`test phenotype.
`
`20
`
`In some embodiments,
`
`the population of cells is selected using a
`
`selection that is lethal to cells which do not display the test
`
`phenotype. Cells which survive the selection therefore display the
`
`test phenotype and form the selected cell population,
`
`
`
`25
`
`In other embodiments, cells displaying the test phenotype may be
`
`isolated and/or separated from other cells in the library to form the
`
`selected cell population.
`
`
`Another aspect of the invention provides a method of genomic screening
`
`30
`
`comprising;
`
`
`providing a library of mutant mammalian cells, each cell
`
`in the
`
`library expressing an RNA-guided endonuclease and a gRNA specific for
`
`a target gene,
`
`such that the target gene is inactivated in the cell,
`
`wherein the library expresses gRNA molecules specific for a set
` of target genes, such that a gene from the set of target genes is
`
`35
`
`inactivated in each mutant mammalian cell in the library,
`
`selecting a population of cells from the library that display a
`
`test phenotype,
`
`

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`WO 2015/040075
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`PCT/EP2014/069825
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`determining the amounts of nucleic acid sequences that encode
`
`GRNA molecules in the selected cell population relative to a control
`
`sample of the library, and;
`
`
`
`identifying one or more nucleic acid sequences encoding gRNA
`
`molecules that are amplified or depleted in the selected cell
`
`population relative to the control sample, and
`
`identifying from the one or more identified nucleic acid
`
`sequences one or more target genes that are inactivated in the
`
`selected cell population.
`
`Preferably, each cell in the library comprises a nucleic acid encoding
`
`an RNA-guided endonuclease and a nucleic acid encoding a gRNA specific
`
`for a target gene stably integrated into the genome of the cell. The
`
`
`
`nucleic acids expressing the RNA-guided endonuclease and gRNAs may be
`
`stably or conditionally expressed in the library of cells in order to
`
`am
`
`10
`
`15
`
`inactivate the set of target genes in the library.
`
`
`Methods of the invention may be useful in identifying genes that
`
`modulate or are functionally linked to the test phenotype in the
`
`
`
`mammalian cell.
`
`A suitable library of mutant mammalian cells may be produced by a
`
`method comprising;
`
`providing a population of mammalian cells that express a RNA
`
`guided endonuclease
`
`providing a population of integrative vectors, each integrative
`
`vector comprising a nucleic acid sequence encoding a guide RNA
`
`molecule (gRNA)
`
`that is specific for a target gene in the mammalian
`
`cells, wherein said population comprises nucleic acid sequences
`
`30
`
`encoding a diverse population of guide RNA molecules (gRNAs)
`
`that is
`
`specific for a set of target genes within the mammalian cell,
`
`transfecting or infecting the mammalian cells with the population
` of integrative vectors such that one or more integrative vectors
`
`stably integrate into the genome of each of the cells,
`
`thereby producing a library of mutant cells that express a RNA-
`
`guided endonuclease and a member of the diverse population of gRNAs,
`
`such that a gene from the set of target genes is inactivated in
`
`each mutant mammalian cell in the Library.
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`

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`WO 2015/040075
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`The mammalian cells may comprise a nucleic acid encoding a RNA-guided
`
`endonuclease integrated into the genome of the cells.
`
`Preferably,
`
`the RNA-guided endonuclease is Clustered Regularly
`
`Interspaced Short Palindromic Repeat
`
`(CRISPR)-associated 9
`
`(Cas9)
`
`nuclease.
`
`Another aspect of the invention provides a diverse population of
`
`integrative vectors,
`
`each integrative vector being capable of integration into the
`
`genome of a mammalian cell and comprising a nucleic acid sequence
`
`encoding a guide RNA molecule (gRNA)
`
`that is specific for a target
`
`gene in the mammalian cell,
`
`
`
`10
`
`15
`
`
`
`wherein said population of vectors comprises nucleic acid
`
`sequences that encode a diverse population of guide RNA molecules
`
`(GRNAS)
`
`that is specific for a set of target genes within the
`
`mammalian cell.
`
`Preferably,
`
`the integrative vectors are viral vectors, most preferably
`
`20
`
`lentiviral vectors.
`
`
`Diverse populations of integrative vectors may be useful
`
`in the
`
`methods described above.
`
`25
`
`30
`
`35
`
`Another aspect of the invention provides a library of mutant mammalian
`
`cells,
`
`each mutant mammalian cell in the library expressing an RNA-
`
`guided endonuclease and a guide RNA molecule specific for target gene,
`
`such that the target gene is inactivated in the cell,
`
`wherein the library expresses a diverse population of gRNA
`Cc
`
`
`molecules that is specific for a set of target genes,
`
`such that a gene
`
`from the set of target genes is inactivated in each mutant mammalian
`
`cell in the library.
`
`Preferably, each mammalian cell in the library comprises a nucleic
`
`acid encoding RNA-guided endonuclease and a nucleic acid encoding a
`
`gGRNA specific for a target gene integrated into the genome thereof.
`
`

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`WO 2015/040075
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`PCT/EP2014/069825
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`
`The set of target genes may comprise all of the protein coding genes
`
`in the genome of the mammalian cell (i.e.
`
`
` library) or a subset of the genes in the genome of the mammalian cell.
`
`the library is a pan-genomic
`
`Populations of mammalian cells may be useful
`
`in the methods described
`
`above.
`
`
` Brief Description of Figures
`
`Figure 1 is a schematic of the gRNA cloning vector. BbsI digestion
`
`10
`
`removes the spacer and produces cohesive ends, which allows duplex
`
`
`
`oligos with compatible overhangs to ligate into. Underlined, BbsI
`
`sites. For the duplex oligos, G at the +l position is highlighted in
`
`red.
`
`gRNA sequences are shown as N
`
`(top strand) and n (bottom strand).
`
`Figure 2 shows the sequences and the genomic positions of gRNAs
`
`targeting the Piga gene. The genomic PAM sequences of each gRNA are
`
`shown in red.
`
`
`
`Figure 3 shows a summary of flow cytometry analysis of GPI-anchored
`
`
`protein expression performed 6 days post transfection. Transfection
`
`
`was performed in triplicates. Data are shown as mean + s.d.
`
`
`
`Figure 4 is a schematic of the piggyBac vector carrying human EFla
`
`promoter-driven puromycin resistant gene (Puro) and humanized Cas9
`
`(hCas93). The two coding sequences are fused with the T2A self-cleaving
`
`peptide. PB, piggyBac repeats; bpA, bovine polyadenylation signal
`
`sequence.
`
`
`Figures 5 and 6 show flow cytometry analyses of GPI-anchored protein
`
`expression (Fig 5) and GFP (Fig 6) after transient transfection. The
`
`parental wild type ESCs
`
`transfected with the indicated plasmid DNAs. The expression of GPI-
`
`anchored proteins was analysed 6 days post transfection by FLAER
`
`(JM8) and the hCas9-expressing clones were
`
`staining (Fig 5). pBluescript was used as a carrier plasmid. To
`
`
`
`measure transfection efficiency,
`
`the celis were also transfected
`
`Separately with a GFP plasmid and analysed 2 days post transfection
`
`(Fig 6).
`
`20
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`25
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`35
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`WO 2015/040075
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`PCT/EP2014/069825
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`Figure 7 is a schematic of the piggyBac vector carrying the gRNA
`
`expression cassette and a neomycin resistant gene cassette. U6, human
`
`U6 promoter; T7, U6 terminator; PGK, mouse Pgki promoter.
`
`
`
`Figure 8 shows flow cytometry analysis of doubly transgenic mouse ESC
`
`lines. This analysis was performed 3 days after the colonies were
`
`picked.
`
`Figure 9 shows histograms showing the distribution of indel sizes at
`
`the on-target locus. Data for parental ESCs
`
`(JM8) and two doubly
`
`transgenic lines (5-4 and 8-3) are shown.
`
`Figure 10 shows histograms the distribution of indel sizes at the off-
`
`target locus 120-tm5-21. Data for parental ESCs
`
`(JM8) and two doubly
`
`transgenic lines (5-4 and 8-3) are shown.
`
`Figure 11 shows histograms the distribution of indel sizes at the off-
`
`target locus 120-tm5-2. Data for parental ESCs
`
`(JM8) and two doubly
`
`transgenic lines (5-4 and 8-3) are shown.
`
`Figure 12 shows a schematic of the self-inactivating lentiviral vector
`
`that expresses gRNA. The FPgkl promoter-driven puro-2A-BFP cassette was
`
`
`inserted downstream of the gRNA expression cassette.
`The
`sequences
`
`shown are the gRNAs targeting Site 3 of the Piga gene with the
`
`endogenous sequences (top) or the modified sequences
`
`(bottom). Note
`
`that the +1 position of the U6 transcript has been changed to a
`
`
`
`G nucleotide in the modified sequence.
`
`CMV, CMV promoter; RU5, 5’
`
`long terminal repeat lacking the U3 region; U6, human U6 promoter; T7,
`
`U6 terminator; PGK, mouse Pgkl promoter; BFP, blue fluorescent
`
`protein; 2A, self-cleavage peptide; puro, puromycin resistant gene;
`
`AU3RU5, enhancer-deleted 3’ LTR.
`
`Figure 13 shows the inactivation of the Piga gene by lentiviral
`
`10
`
`15
`
`20
`
`25
`
`30
`
`delivery of the gRNA expression cassette in hCas9-expressing mouse
`
`35
`
`ESCs
`
`(upper) or mouse pancreatic carcinoma cells (lower). Cells were
`
`
`
`infected with lentivirus expressing the gRNA targeting site 3 of the
`
`Piga gene. Transduced cells were analysed by flow cytometry 6 days
`
`post infection. While cells transduced with the empty lentivirus were
`
`
`
`

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`WO 2015/040075
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`PCT/EP2014/069825
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`all FLAER-positive, cells transduced with Piga:gRNA-expressing
`
`lentivirus were generally FPLAFR-negative population.
`
`Figure 14 shows the inactivation of the Piga gene by lentiviral
`
`delivery of the gRNA expression cassette in hCas9-expressing mouse
`
`ESCs infected with lentivirus expressing the gRNA targeting the
`
`indicated sites of the Piga gene with the endogenous
`
`(top) or altered
`
`(bottom) sequences. Transduced cells were analysed by flow cytometry 6
`
`days post infection.
`
`
`Figure 15 shows a summary of the cytometry analysis shown in figure
`
`14. Data are shown as mean + s.d.
`
`(n=2). Student’s t-test was
`
`
`
`performed.* p<0.01
`
`Figure 16 to 19 ahow analyses of 52 gRNAs targeting the 26 genes
`
`involved in the GPI-anchor biosynthesis pathway.
`
`
`Figure 16 shows the fractions of reads with indels analysed by deep
`
`sequencing. **, no indel was detected.
`
`Figure 17 shows flow cytometry analysis of cells transfected with the
`
`indicated gRNA-expression vectors. pBluescript was used as a control
`
`vector. *, not significant when compared to the control by Student’s
`t-test.
`
`10
`
`15
`
`20
`
`25
`
`Figures 18 and 19 show the percentage of in-frame indels. The y axis
`
`shows the number of reads with in-frame indels at the indicated size
`
`
`divided by the total numbers of reads with all indels at Site 1
`
`
`
`(Figure 18) and at Site 2
`
`(Figure 19). **, no indel was detected. Data
`
`30
`
`are shown as mean + s.d.
`
`(n=2-4).
`
`Figure 20 shows a schematic of genetic screens with genome-wide
`
`lentiviral gRNA libraries.
`
`Figure 21 shows gRNA design statistics (far left) and deep sequencing
`
`analyses of the gRNAs
`
`in the lentiviral plasmid DNA library (centre
`
`(far right).
`
`(centre right) and 2
`
`left), ESC library 1
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`

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`WO 2015/040075
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`Figure 22 shows scatter plots comparing gRNA frequencies in the
`
`original lentiviral plasmid DNA and in the ESC library 1
`
`(1H) and 2?
`
`
`
`(RH).
`
`gGRNA counts in the ESC libraries have been normalised against
`
`the gRNA counts from tne lentiviral library.
`
`Figure 23 shows fold changes of read counts between the lentiviral
`
`plasmid DNA library and the ESC libraries. Pluripotency genes, Nanog
`
`and Pou5fl, and essential genes, Rad5i and Breal, are significantly
`
`depleted in the ESC libraries, whereas lineage marker genes are not
`
`depleted. The same gRNAs
`
`in the two ESC libraries are linked with
`
`lines. Mann-Whitney U test was performed by comparing gRNAs of each
`
`
`in the corresponding ESC
`gene from each library with all gRNAs
`
`library.
`
`Figures 24 and 25 show genetic screens using the genome-wide gRNA
`
`library and genetic validation assays of the novel candidate genes.
`
`
`
`Genes with multiple gRNA hits in cells resistant to alpha-toxin are
`
`shown in Fig 24 and 6TG in Fig 25. All known genes involved in the
`
`GPI-anchor synthesis pathway are shown in Fig 24. Genes highlighted in
`
`asterisk were chosen for further validation. MMR, mismatch repair.
`
`10
`
`15
`
`20
`
`
`
`
`
`Figures 26 and 27 show the enrichment of gRNA sequences after
`
`treatment with either alpha-toxin (Fig 26) or 6TG (Fig 27)
`
`treatment.
`
`Four independent mutant cell libraries were used in each treatment.
`
`*o<0.01, **p<0.001 by T-test.
`
`Figure 28 shows a summary of the guide RNA hits in the 26 GPI-anchor
`
`pathway genes identified in Example 2.10.
`
`30
`
`Table 1 shows indel patterns on the on-target sites in the doubly
`
`transgenic colonies. Sequences in red and green represents the PAM and
`
`GRNA sequences, respectively. Mismatch bases are shown in blue. The
`
`Sizes of the deletions were shown on the right. Note that colony 8-3
`
`and 8-9 carry multiple numbers of deletions.
`
`Table 2 shows a summary of gRNA hits in genome-wide screens.
`
`

`

`WO 2015/040075
`
`
`Detailed Description
`
`PCT/EP2014/069825
`
`The methods described herein relate to genomic screens to identify
`
`genes and other genomic sequences that modulate, support or are
` functionally linked to a phenotype of interest.
`
`Screening methods described herein employ libraries, collections or
`
`pools of mutant mammalian cells in which different members of a set of
`
`target genes are selectively inactivated or “knocked out” in different
`
`cells in the library. The target gene is inactivated or knocked out
`
`in
`
`
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`the cells of the library through the expression from integrated
`c
`genomic nucleic acid sequences of
`
`as Clustered Regularly Interspaced Short Palindromic Repeat
`
`i) an RNA guided endonuclease,
`
`(CRISPR)-
`
`such
`
`associated 9
`
`(Cas93) nuclease, and ii) a gRNA molecule that directs the
`
`endonuclease to the specific
`
`target gene. The RNA guided endonuclease
`
`and the gRNA molecule together cause double strand cleavage of the
`
`genomic DNA in both alleles of the target gene. Repair of these DNA
`
`double strand breaks (DSBs) by the mammalian cell leads to the
`
`
`
`introduction of insertion or deletion (indel) mutations that
`
`inactivate the target gene.
`
`llargeted gene inactivation using gRNA and RNA guided endonucleases
`
`that are stably integrated into a cell genome provides stable
`
`expression over time in the Library of cells and has not been reported
`
`previously. Furthermore,
`
`the amount of gRNA and RNA guided
`
`endonuclease expression from stably integrated coding sequences is
`
`
`shown herein to be sufficient to mediate targeted DNA double strand
`
`breakage and gene inactivation.
`
`
`
`
`Different members of the library of mutant mammalian cells express
`
`different gRNA molecules specific for different target genes,
`
`such
`
`that different genes from the set of target genes are inactivated in
`
`different mutant mammalian cells in the library.
`
`The library of mutant mammalian cells may be used inter alia for loss-
`
`of-function screening. The library may be subjected to positive or
`
`negative selection for a test phenotype,
`
`such as drug resistance, and
`
`nucleic acid sequences encoding gRNA molecules may be identified in
`
`the selected cell population that displays the phenotype. From these
`
`

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`WO 2015/040075
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`10
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`PCT/EP2014/069825
`
`sequences,
`
`identified. Optionally,
`
`target genes that mediate the test phenotype may be
`
`the amounts or numbers of copies of one or
`
`more nucleic acid sequences that encode gRNA molecules may be
`
`determined in the selected cell population. The most abundant gRNA
`
`encoding nucleic acid sequences in the selected cell population, or
`
`threshold amount
`
`the gRNA encoding nucleic acid sequences that are present above a
`
`in the selected cell population may be identified.
`
`Target genes that mediate the phenotype may be identified from these
`
`
`
`identified gRNA encoding nucleic acids.
`
`10
`
`In some embodiments,
`
`the library may be subjected to selection for
`
`
`cells having a specific phenotype,
`
`such as drug resistance or a
`
`cellular response to a stimulus or chemical compound, and the amounts
`
`of nucleic acids encoding different gRNA molecules in the population
`
`of selected cells (i.e.
`
`in cells which display the test phenotype) may
`
`
`
`be determined relative to a control
`
`sample of the library (e.g.
`
`a
`
`library sample that has not been subjected to selection).
`
`encoding nucleic acid sequences that are amplified or depleted in the
`
`gRNA
`
`20
`
`population of selected cells relative to the control sample may be
`
`identified and used to identify target genes that mediate the
`
`phenotype.
`
`Genes which are targeted by the gRNAs encoded by nucleic acids that
`
`are enriched or depleted may be identified from the recognition
`
`sequences of the enriched or depleted gqRNA-encoding nucleic acids.
`
`The target genes identified by the methods described herein are
`
`associated with or involved in the phenotype that is tested.
`
`For
`
`example,
`
`the identified genes may modulate, mediate or be functionally
`
`30
`
`linked to the selected phenotype in the mammalian cells or may be
`
`activators or repressors of a phenotypic cellular response.
`
` Because the gRNA-encoding nucleic acid is integrated into the genome
`of the mammalian cell, each cell in the library carries retrievable
`
`information which identifies the gene that is inactivated in it. This
`
`internal tag allows the screening methods described herein to be
`
`performed on a library or pool of mammalian cells in a single step.
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`This avoids the need for large-scale parallel testing of individual
`
`clonal cell populations with known deletions (i.e. an array format).
`
`Because both alleles of the target genes in the library of mutant
`
`cells are inactivated by the RNA guided endonuclease, both recessive
`
`and dominant genes may be identified by the methods described herein.
`
`Methods of the invention are performed in vitro.
`
`10
`
`The mammalian cells are preferably isolated cells and may be from any
`
`human or non-human mammalian species, preferably human or mouse cells.
`
`Suitable mammalian cells for use in the methods described herein
`
`include somatic cells, pluripotent cells, somatic stem cells and
`
`15
`
`cancer cells.
`
`Mammalian pluripotent cells may include embryonic stem (ES) cells,
`
`for
`
`example murine ES cells, and non-embryonic stem cells,
`
`including
`
`foetal and adult somatic stem cells and stem cells derived from non-
`
`20
`
`pluripotent cells, such as induced pluripotent
`
`(1PS) cells.
`
`The mammalian cells may be from an established cell line,
`
`for example,
`
`a cancer cell line such as human lung carcinoma cell line A549, or may
`
`be obtained from a cell sample from an individual,
`
`for example a human
`
`25
`
`or non-human mammal.
`
`Preferably,
`
` cells are well-known in the art.
`
`the mammalian cells are dividing cells. Suitable mammalian
`
`30
`
`The cells in the mutant mammalian cell library express a RNA guided
`
`endonuclease and a gRNA. Preferably,
`
`cells. The RNA guided endonuclease forms a complex with the gRNA and
`
`the expression is stable in the
`
`cleaves both strands of the target gene at a DNA scquence (termed a
`
`recognition region or protospacer) with the target gene that is
`
`complementary to the target sequence (or crRNA region) of the gRNA.
`
`
`The nucleic acid encoding the RNA guided endonuclease may for example
`
`be stably integrated into the genome of the mammalian cells at a
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`neutral and active site in the genome , such as the mouse Rosa26 lL
`
`or its human homologue (Irion et al Nature Biotech 25 (12) 1477-1482),
`the AAVS] site (Sadelain, M. et al. 2011 Nat Rev Cancer.
`(2011)
`I;
`
`Oo
`
`12(1):51-8) or the Mouse Collal
`
`locus (Beard, C. et al. Genesis 44:
`
`23-28) or its human homologue.
`
`In preferred embodiments,
`
`the RNA guided endonuclease is Clustered
`
`Regularly Interspaced Short Palindromic Repeat
`
`(CRISPR)-associated 9
`
`(Cas9) nuclease,
`
`The clustered regularly interspaced short palindromic repeat
`(CRISPR) /CRISPR associated protein (Cas)
`system is an adaptive immune
`mechanism found in bacterial and archaeal species that allows the host
`
`to combat pathogens, such as bacteriophages (Barrangou, R. et al.
`Science 315, 1709-1712 (2007); Marraffini, L.A.
`& Sontheimer, E.J.
`
`Science 322, 1843-1845 (2008);
`Bhaya, D., Davison, M.
`& Barrangou, R.
`Annual review of genetics 45, 273-297 (2011); Garneau, J.E. et al.
`
`Nature 468, 67-71 (2010)). Bacteriophage-derived 30-bp DNA fragments
`are inserted into the CRISPR locus of the host cell and transcribed as
`
`CRISPR RNAS
`
`(crRNAs). These form a complex with trans-encoded RNA
`
`{tracrRNA) and CRISPR-associated (Cas) proteins, and the complex
`introduces site-specific cleavage at DNA sites that match the sequence
`of the crRNAs.
`
`
`
`The use of the CRISPR/Cas system to introduce site-specific DNA DSBs
`into cultured mammalian cells has been reported in the art
`(Jinek, M.
`
`et al. Science 337, 816-821 (2012); Gasiunas, G., Barrangou, R.,
`
`Horvath, P.
`& Siksnys, V. Proc. Natl. Acad. Sci. USA 109, E2579-2586
`
`(2012); Cong, L. et al. Science 339, 819-823 (2013); Mali, P. et al.
`
`Science 339, 823-826 (2013); Cho, S.W., Kim, S., Kim,
`
`J.M.
`
`& Kim, J.S.
`
`Nature Biotechnology 31, 230-232 (2013); Wang, H. et al. Cell 153,
`
`910-918 (2013)).
`
`the celis in the mutant mammalian cell library express a
`Preferably,
`
`Cas9 nuclease and a gRNA molecule. The Cas9 nuclease forms a complex
`with the gRNA molecule and introduces a site-specific DNA double
`
`strand break into DNA sequences (termed recognition regions or
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`290
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`protospacers)
`
`region) of the gRNA molecule.
`
`that are complementary to the target sequence (or crRNA
`
`
`The amino acid sequences and encoding nucleic acid sequences of
`
`Suitable Cas? nucleases for use as described herein are well-known in
`
`
`=
`the art. Suitable Cas9 nuclease sequences include SEQ ID NO: 1.
`
`Suitable Cas9 nucleases for use as described herein may be derived
`
`
`
`from Streptococcus pyogenes SF370, Streptococcus thermophilus LMD-9
`
`(Cong, L. et al. Science 339, 819-823 (2013)) or other bacterial or
`
`10
`
`archeal species.
`
`The nucleic acid sequence encoding the Cas9 nuclease or other RNA
`
`guided endonuclease may be humanized,
`
`i.e. codon-optimised for
`
`expression in human cells.
`
`15
`
`Nucleic acid encoding Cas9 nucleases and other RNA guided
`
`endonucleases may be produced by conventional synthetic means or
`
`obtained from commercial suppliers (System Biosciences, USA; BioCat
`
`
`GmbH, DE) or non-profit repositories (e.g. Addgene, MA USA).
`
`20
`
`In some embodiments,
`
`the amino acid sequences of suitable RNA guided
`
`endonucleases may be fused to nuclear translocalisation signals (NISs)
`
`
`
`and/or tags. Suitable NLSs and/or tags and encoding nucleic acids are
`
`well-known in the art. For example,
`
`a Cas9 nuclease may be fused at
`
`one end or both ends to a nuclear localisation signal and/or a
`
`sequence tag,
`
`such as a FLAG, HA, Myc, V5 tag.
`
`The nucleic acid sequence encoding the Cas9 nuclease or other RNA
`
`guided endonuclease may be operably Linked to a suitable regulatory
`
`element. Suitable regulatory elements are active after stable
`
`integration into the mammalian genome and include constitutive
`
`promoters, such as human elongation factor la (EFla) promoter, CAG
`
`promoter, human ubiquitin C promoter, human/mouse PGK promoter, and
`
`human/mouse PolII promoter; and conditional promoters, such as the
`
`tetracycline response element
`
`(TRE) promoter.
`
`30
`
`35
`
`The nucleic acid sequence encoding the Cas9 nuclease (i.e. Cas9
`
`nucleic acid) or other RNA guided endonuclease may be contained in an
`
`expression vector. Expression vectors suitable for stable integration
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`WO 2015/040075
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`into the mammalian cell genome and the expression of recombinant
`£
`proteins are well known in the art.
`
` Suitable vectors can be chosen or constructed, containing appropriate
`
`regulatory sequences,
`
`including promoter sequences,
`
`terminator
`
`fragments, polyadenylation sequences, enhancer sequences, marker genes
`
`and other sequences as appropriate. Preferably,
`
`the vector contains
`
`appropriate regulatory sequences to drive the expression of the Cas9
`
`or other RNA guided endonuclease in the mammalian cells.
`
`
`For ease of manipulation,
`
`a vector may also comprise sequences, such
`
`as origins of replication and selectable markers, which allow for its
`
`selection and replication and expression in bacterial hosts such as E£.
`
`coli as well as in mammalian cells.
`
`10
`
`15
`
`Vectors suitable for use in expressing RNA guided endonuclease
`
`encoding nucleic acids include plasmids and viral vectors e.g.
`
`'phage,
`
`or phagemid, and the precise choice of vector will depend on the
`
`20
`
`particular expression system which is employed. Preferably,
`
`
`for example,
`is an integrative vector. For further details see,
`
`the vector
`
`Molecular Cloning: a Laboratory Manual: 4th edition, Green et al.,
`
`2012, Cold Spring Harbor Laboratory Press.
`
`Nh Oo
`
`30
`
`35
`
`
`
`In preferred embodiments,
`
`the expression vector comprising the nucleic
`
`acid encoding the Cas9 nuclease or other RNA guided endonuclease
`
`stably integrates into the genome of the mammalian cells following
`
`transfection. Each mammalian cell may contain one copy or multiple
`
`copies of the nucleic acid sequence encoding the RNA guided
`endonuclease.
`
`Tn some embodiments,
`
`the nucleic acid encoding the RNA guided
`
`
`
`endonuclease may be stably integrated into the genome of the mammalian
`
`cells at a neutral and active site in the genome,
`
`such as the mouse
`
`RosaZ26 locus or its human homologue (Irion et al Nature Biotech 25
`
`(12) 1477-1482} or the mouse Collal
`
`locus or its human homologue.
`
`
`
`Mammalian cells may be transfected or infected with suitable
`
`expression vectors for stable integration of the nucleic acid encoding
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`the RNA guided endonuclease using standard recombinant techniques.
`
`Following transfection,
`
`the RNA guided endonuclease may be
`
`constitutively or conditionally expressed in the mammalian cells.
`
`transfection and gene
`
`Techniques and protocels for transformation,
`
`expression in cell culture are well known in the art
`
`Protocols in Molecular Biology, Second Edition, Ausubel et al. eds.
`
`John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS
`
`(see for example
`
`
`
`Tuan
`
`(Mar 1997} Humana Press Inc).
`
`In some embodiments,
`
`transfected cells that express the RNA guided
`
`endonuclease may be selected following transfection,
`
`for example using
`
`
`
`
`
`a selectable marker such as antibiotic resistance or fluorescence,
`
`such that the transfected cells are isolated from cells that do not
`
`express the RNA guided endonuclease.
`
`In other embodiments, separation of transfected cells that express the
`
`RNA guided endonuclease from cells that do not express the RNA guided
`
`endonuclease may not be