Efficient generation of conditional knockout mice by CLICK

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1 Efficient generation of conditional knockout mice by CLICK Tomoji Mashimo Genome Editing Research and Development (R&D) Center and Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University

2 Generation of Knockout (KO) animals by CRISPR/Cas Cas9 mrna/grna CAP CAP AAA AAA G A Male pronucleus (100/50 ng/μl) Microinjection G A transfer Pseudopregnant mother deliver Pups (Funders) Advantage Knockout mice can be generated in any background strain within 2 4 months (ES cells: months) Multiple genes are modified by co injection of several grnas Co injection of donor DNA with homology arms can provide knock in mice Genome editing is available in other species,rat,pig,monkey (Mashimo T, Dev Growth Differ. 2014)

3 Knockout by CRIPR/Cas system CRISPR/Cas9 transfer Wistar #1 8,#4 3(Ho) #2 4(Ho),#2 6 #5 3,#6 6 #2 8,#4 6(Ho) #2 8 #3 7(Ho),#3 8(Ho) #6 4(Ho) #6 2,#6 6,#6 7(Ho) #6 2 #6 3(Ho) CATGGTTTCCAGGATTATGTAATAGTGGTCCCT CATGGTTTCCAGGATTATGTAATTAGTGGTCCCT CATGGTTTCCAGGATTATGTAAATAGTGGTCCCT TTTGT /------CATCA CATGGTTTCCAGGATTATGTAA-/----TAATA CATGGTTTCCAGGAT TAGTGGTCCCT CATGGTTTCCAGGATTATGTAAAATAGTGGTCCCT CATGGTTTCCAGGATTATGTAAGATAGTGGTCCCT CATGGTTTCCAGGATTAT---TTAGTGGTCCCT Injected mrna Injected embryos Collected cells (%) PCR-amplified (%) KO- Mutations (%) Generating KO rats is possible with CRISPR! grna + Cas (45.6) 34 (82.9) 14 (41.2)

4 Allele-specific genome editing and correction of diseaseassociated phenotypes in rats using CRISPR/Cas platform (KYoshimi et al. Nat Commun 2014 Jun 26;5:4240.) Efficient knock insofsmalldnafragmentsinzygotes

5 Genome engineering by zygote electroporation grna + Cas9 (+ ssodn) grna + Cas9 polya + ssodn Target locus Embryos injected Two cell embryos (%) Pups delivered (%) KO (%) KI/offspring (%) Il2rg (90.6) 32 (55.2) 3 (9.4) Il2rg (75.0) 24 (53.3) 21 (87.5) 8 (33.3) NEPA21 electroporator Easy transfer (without special skills) 5 min and low toxicity KO and KI with ssodn High throughput engineering

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7 Genome engineering by zygote electroporation Gene Knockouts by ZFN, TALEN, CRISPR! Gene Knock ins by CRISPR with ssodn! (Kaneko et al, Sci Rep. 2014) (Kaneko et al, Sci Rep. 2015)

8 Knock in technologies with CRISPR/Cas Electroporation mrna Microinjection CAP AAA CAP AAA CAP CAP + AAA AAA Transfer (Kaneko et al. Donor Sci Rep DNA 2014) Pseudopregnant rats Founder pups ssodns donor Targeting vector Homology directed repair Possible (HDR) Zygote Homologous recombination DSB repair electroporation Impossible (HR) High Custom DNA (ssodn) Efficiency Prepare Low Construct homology arms Short (<50bp) Insert size Long (<10kb) New technology for large DNA Knock in is needed!

9 Protocol to make long single-stranded DNA(lssDNA) targeting sequences nicking site nicking site double-stranded DNA plasmids Nicking endnucleases (1 hour) Double nicking methods Nick Nick plasmid Gel extraction (1 hour) Electrophoresis (1 hour) Simple! ~3 kbp single-strand oligo (LssDNA) can be prepared by easy protocols (Yoshimi Nat Commun 2016)

10 Long ssodn(lssdna) can be used for GFP-KIs Cas9 mrna CAP AAAA CAP AAAA grna lsodn Transfer Deliver #1 #3 #2 PCR, Sequencing 1-hit 1-oligo (1H1O) by long ssodn (lsodn) rat Thy1 F2 lsodn-atg(+) GFP 5 3 TGAACCCAGTCA TCAGCA TC grna:thy1-atg exon 2 3 R2 F1 F3 ATG TGA R1 4 grna:thy1-tga GGACTTCATTTCTCTGTGAC 3 5 R3 2AGFP lsodn-tga(-) F4 R4 Wistar lsodn TGA( ) #7 #9 #10 KI Thy1-TGA target PAM GCCACGGACTTCATTTCTCTGTGACTGGTTGGG CTCTGAGG-CCAATG---TAACTG-AACTGACTGGTG HA (60bp) 2A (69bp) GFP (720bp) 3 UTR (45bp) HA (300bp) KI/KO CTCTGAGG-CCAATG---TAACTG-AACTGACTGGTG KI/KO CTCTGAGG-CCAATG---TAACTG-AACTGACTGGTG CTCTGAGG-CCAATG---TAACTG-AACTGACTGGTG #11 KI CTCTGAGG-CCAATG #18 KI TAACTG-AACTGACTGGTG #24 KI CTCTGAGG-CCAATG---TAACTG-AACTGACTGGTG 11.1% (4/36 knock-in/total offspring) high efficiency (Yoshimi Nat Commun 2016) cortex cerebellum

11 ssodn-mediated knock-in with CRISPR/Cas for large genomic regions in zygotes (KYoshimi et al. Nat Commun 2016 Jan 20;7: ) HP: u.ac.jp/ja Efficient knock in for large DNA fragments in zygotes

12 CAG GFP Knock in at Rosa26 locus by 2H2OP 1 st STEP rat Rosa26 grna:rrosa26 CGT GATCTGCAACTGGAGTC exon1 2 F1 R1 grna:caggs CAGGGTTATTGTCTCATGAG pcag-gfp F3 CAG R3 GFP pa 2 nd STEP Primer Rosa26 GFP Rosa F CAG R CAG F Rosa R Injected RNA and DNA F1 ssodn-1 CAG R1 R3 grna ssodn plasmid rrosa26, pcag rrosa pcag, pcag rrosa F2 GFP pa R2 Embryos injected pcag GFP 119 F3 ssodn-2 W Two cell Embryos (%) 66 (55.5) A E C D C D A E 11 C D TCTTCCCTCGTGATCTGCAACTGGAGTCTTTCT ATTTATCAGGGTTATTGTCTCATGAGCGGATACA TCTTCCC------GCGGATACA TCAGGGTTATTGTCTCATGATCTGCAACTGGAGTCT GGCGG-( 256bp)-GAGCGGATACA TTATTGTCTCA-GTC(+30bp)TAC--TCTGCAACT GCCTG----( 506bp)----TTGTA ATTTATCAGGGTTATTGTCTCATGAGCGGATACA TCTTCCCTCGTGAGCGGATACA TCAGGGTTATTGTCTCATGATCTGCAACTGGAGTCT Pups Delivered (%) 17 (25.8) Knockout (%) 15 (88.2) Knock in (%) 3 (17.6) KI phenotype (n, %) GFP positive (n=4, 23.5%)

13 Human BAC (200 kb) Knock in by 2H2OP 1 st STEP grna:piseci CTTTCTCCGCACCCGAC ATA F2 grna:rsirpa GTGTCCTCCCTGACGCCTGT exon 1 2 R2 F1 rat Sirpa R human SIRPA (45-kbp) kbp RP11-933C kbp A (F1-R1) B (F3-R3) C,D # M # M (F1-R2) 2 * * 8 (F2-R1) 2 8 M 2 nd STEP W 2 A E C D GCACTGTGTCCTCCCTGACGCCTGT GGGACCCA TTATCTATGTCGGGTGCGGAGAAAG AGGTAATG GCACTGTGTCCTCCCTGACGCCAAG AGGTAATG TTATCTATGTCGGGTGCGGAGATGT GGGACCCA rsirpa Knockout & hsirpa Knock-in (humanization) ssodn-1 SB-B F1 F3 SB-A F2 ssodn-2 R2 R3 R1

14 Genome engineering in mouse zygotes Multiplex knockout Knockout Large deletion GeneX GeneY GeneX GeneZ Target Conditional (flox) NHEJ repair DSB ssodn (80 120bp) HR repair Cassette integration GFP LoxP ssodns Knock-in LoxP ssodns GFP

15 Conditional knockout mice by CRISPR/Cas9! How can you knock in two loxp at one allele? (CRISPR with lssdna inserts conditional knockout alleles)

16 Floxed alleles generated by CLICK conventional ES loxp exon exon loxp vector CRISPR DSB exon DSB ssodn ssodn Novel method CLICK DSB exon exon DSB lssdna

17 CLICK: CRISPR with lssdna inducing conditional knockout alleles

18 Flox rats generated by CLICK! Genotyping PCR, sequencing Primer set 1: Primer set 2: exon SNP exon * No. 識別 ( 耳 ) 性別 Exon deletion LoxP KI Up 遺伝子解析 LoxP KI Down SNP mutation 1 M 2 R M 3 L M 4 RL M 5 F 6 R F Homozygous KI of flox alleles!

19 Conditional knockout mice by CLICK! Conventional (1 ~ 2 year) ES ES cells chimera flox Cre CKO CRISPR (ssodn) grna Cas9 ssdna CLICK (3~6 month) *loxp mice KI mice of single loxp site flox Cre CKO CRISPR (lssdna) grna Cas9 lssdna flox Cre CKO Cre-expressing embryos grna Cas9 lssdna CKO

20 Genome engineering in mouse zygotes Multiplex knockout Knockout Large deletion GeneX GeneY GeneX GeneZ Target Conditional (flox) NHEJ repair DSB ssodn (80 120bp) HR repair Cassette integration GEP LoxP ssodns Knock-in LoxP (Miyasaka under review) ssodns GEP

21 Genome engineering in mouse zygote! Knockout ssodn Knock in lssdna HR Knock in 2H2OP Knock in Goal Knockout gene SNP Tag, LoxP ~50 bp Long Plasmid ssodn Flox GFP alleles Cre ~10 ~5Kbp GFP Cre BAC Replace ~200 Kbp Repair NHEJ SDSA SDSA HR NHEJ +SDSA Efficiency 60 90% 20 40% 10 20% 10 30% 0 10% 5 30% Advantage Efficient Multiple Any strain Injection? Electroporation Efficient GWAS SNP Zygotes flox? Precise Precise Target Simple gene Simple (Kaneko Low Efficient efficient et Time al. Sci Rep Size 2014)? Simple Large DNA Precise? Yoshimi 2016 Nat Commun

22 Severe combined immunodeficiency (SCID) Rats & Rabbits Rag2 & Il2rg KO rats Foxn1 or Il2rg KO rabbits (-/-) (+/+) SCID F344 NBRP Rat u.ac.jp/nbr FOXN1_KO_F0 12 KITAYAMA LABES CO.,LTD. Human cell or tissue xenograft models (Humanization)!

23 Humanized rats Humanized animals: Mice carrying human cells, tissues, and/or organs. Immunodeficient animals are often used as recipients. ips cells Tumor cells FSG rats Hepatocytes teratoma 6 8w Tumor subcutaneous hepatocytes Portal vein testis 2 4w 6 10w SCID FSG Endoderm Ectoderm Mesoderm Teratoma Assay! Tumor bearing! Liver humanized! (Mashimo et al. Cell Reports, 13 Sep 2012)

24 大阪大学大学院医学系研究科共同研附属ゲノム編集センター Genome Editing Research and Development Center (GERDC) launched in 14 th December u.ac.jp/gerdc/

25 Acknowledge