CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus
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1 CRISPR/Cs9: An inexpensive, efficient loss of function tool to screen humn disese genes in Xenopus Frgment nlysis to test the efficcy of CRISPR Cs9 protein is more effective nd less toxic CRISPR/Cs9 provides similr results to morpholino
2 PR Cori Prsk nd Ry Scheid
3 PR Genome engineering CRISPR/Cs9 overview Other methods Pper
4 Wht is genome engineering? Strtegies nd techniques for trgeted nd specific modifictions of the genome
5 Wht is CRISPR/Cs9? ultimedi/rchive/00229/annrev _f1_ gif Clustered regulrly interspced short plindromic repets (CRISPR)
6
7 Wht does the nuclese Cs9 look like? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
8 How does the nuclese Cs9 work? = NGG
9 How does the cell repir double strnd breks?
10 Wht kinds of CRISPR chnges cn you mke?
11 Wht were previous gene editing methods? TALENS & ZFNs
12 Wht were previous gene editing methods? TALENS & ZFNs
13 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
14 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
15 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
16 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
17 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
18 How does CRISPR/Cs9 compre with TALEN nd ZFN? Hyongbum K, Jin-Soo K (2014) Nture Rev Genet 15:
19 Wht is Frgment nlysis (FA)?
20 Wht is Frgment nlysis (FA)?
21 Wht is Frgment nlysis (FA)?
22 How did they knock down genes in Xenopus nd Zebrfish before Cs9? RNA Morpholino oligos (MOs)
23 Xenopus Fst Esy Vertebrtes Diploid Conservtion
24 How is frgment nlysis used to nlyze CRISPR?
25 How is frgment nlysis used to nlyze CRISPR?
26 How is frgment nlysis used to nlyze CRISPR?
27 How is frgment nlysis used to nlyze CRISPR?
28 How do different sgrna s ffect results?
29 How does Cs9 protein toxicity compre to cs9 mrna? 100 A % embryos A 0
30 How does Cs9 protein toxicity compre to cs9 mrna? % embryos 100 A A 0
31 How does Cs9 protein toxicity compre to cs9 mrna? 100 A % embryos A 0
32 How does Cs9 protein toxicity compre to cs9 mrna? % embryos 100 A A 0
33 How does Cs9 protein toxicity compre to cs9 mrna? 100 A % embryos A 0
34 How does Cs9 protein perform compred to cs9 mrna?
35 How does Cs9 protein perform compred to cs9 mrna?
36 How does Cs9 protein perform compred to cs9 mrna?
37 Wht re primry cili? Chemicl senstion Signl trnsduction Control of cell growth
38 Wht re motile cili?
39 How did CRISPR phenotypes compre to morpholino s? Gene/ muttion Foxj1 Ccdc40 Dnh9 OMIM disese phenotype Allergic rhinitis, Autoimmune disese Primry ciliry dyskinesi Primry ciliry dyskinesi Morpholino/mutnt line phenotype 90% decresed epiderml cili Decresed nd bnorml epiderml cili Glnt11 Heterotxy 25% A + L hert loops; incresed epiderml cili Px8 Tyrosinse Kidney genesis, Hypothyroidism Oculocutneous lbinism Bet-ctenin Cncer, intellectul disbility CRISPR phenotyope (% of embryos) 68% decresed epiderml cili 48% decresed nd bnorml epiderml cili 30% lterlity defect 28% A + L hert loops 59% mlformed pronephros Albinism Ventrliztion of embryo 23% A + L hert loops; 50% incresed epiderml cili 40% mlformed pronephros ~100% lbinism No venrliztion
40 How does CRISPRp in foxj1 ffect surfce cili development?
41 How did CRISPR phenotypes compre to morpholino s? Gene/ muttion Foxj1 Ccdc40 Dnh9 OMIM disese phenotype Allergic rhinitis, Autoimmune disese Primry ciliry dyskinesi Primry ciliry dyskinesi Morpholino/mutnt line phenotype 90% decresed epiderml cili Decresed nd bnorml epiderml cili Glnt11 Heterotxy 25% A + L hert loops; incresed epiderml cili Px8 Tyrosinse Kidney genesis, Hypothyroidism Oculocutneous lbinism Bet-ctenin Cncer, intellectul disbility CRISPR phenotyope (% of embryos) 68% decresed epiderml cili 48% decresed nd bnorml epiderml cili 30% lterlity defect 28% A + L hert loops 59% mlformed pronephros Albinism Ventrliztion of embryo 23% A + L hert loops; 50% incresed epiderml cili 40% mlformed pronephros ~100% lbinism No venrliztion
42 How does CRISPRp in px8 ffect kidney development?
43 How did CRISPR phenotypes compre to morpholino s? Gene/ muttion Foxj1 Ccdc40 Dnh9 OMIM disese phenotype Allergic rhinitis, Autoimmune disese Primry ciliry dyskinesi Primry ciliry dyskinesi Morpholino/mutnt line phenotype 90% decresed epiderml cili Decresed nd bnorml epiderml cili Glnt11 Heterotxy 25% A + L hert loops; incresed epiderml cili Px8 Tyrosinse Kidney genesis, Hypothyroidism Oculocutneous lbinism Bet-ctenin Cncer, intellectul disbility CRISPR phenotyope (% of embryos) 68% decresed epiderml cili 48% decresed nd bnorml epiderml cili 30% lterlity defect 28% A + L hert loops 59% mlformed pronephros Albinism Ventrliztion of embryo 23% A + L hert loops; 50% incresed epiderml cili 40% mlformed pronephros ~100% lbinism No ventrliztion
44 Summry Cs9 protein is more effective option thn Cs9 mrna Frgment nlysis provides n effective mechnism to test the efficcy of the CRISPR CRISPR/Cs9 cn reproduce MO phenotypes with fewer cons CRISPR is n efficient screening tool for testing congenitl mlformtion cndidte genes in Xenopus
45 Wht next?
46 Wht next? sgrna ctivity nd efficiency Model orgnisms Specificity Off-trget effects
47 Questions?
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