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CRISPR/Cas9 Gene Editing

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CRISPR/Cas9 Gene Editing Oncology & Oncology & Immunology Immunology In Vivo Target Validation with KO and KI mice • from concept, disease models, to PK-PD-efficacy studies • Generation of cell lines with CRISPR-mediated gene knockout and knockin CRISPR/Cas9 Gene • Genome-wide screening for target identification and validation Timeline for CRISPR/CAS9 based method for knock In Service • Human and mouse CRISPR knockout libraries • Human CRISPR activation libraries 2-4 weeks 2-4 weeks 4-6 weeks 2-3 weeks 3-6 months Editing Platforms • Fast generation of GEMMs for genetic diseases sgRNA design & Microinjection Breeding, In vitro test & QC Genotyping preparation & Transplantation PK PD study F0 ssODN or vector construction (KI) CRISPR/Cas9 for target validation • Target validation in cells or mice in combination with other tools such as lentiviral gene overexpression, RNAi knockdown sgRNA for targeting • Phenotype ssODN for point mutation • Overexpression (Lenti) Engineered • Tumorigenesis • Knockdown (RNAi) cell lines • KO/KI (CRISPR) • Drug sensitivity Donor vector for replacement Target Target engagement In Vivo CRISPR Animal Models • Phenotype • Immune • from genome editing to disease models vs.Tumorigenesis KO/KI (CRISPR) Case study: Leptin and Leptin Receptor Knockout rats for obesity, Case study 2: DEN-induced Aldh2E487K KI mice could accelerate liver tumor GEMM • Tumor formation diabetes and fatty liver development • Drug sensitivity Case study: Gene knockout of gene X in MDA-MB-231 by CRISPR/Cas9 Lep-/- LepR -/- A gRNA #3 B WT WT Control 1 5 6 10 13 Gene X Glucose tolerance test #3-5 #3-1 Parental β-Actin Reference Gene X #3-8 #3-10 g3-6 1. Shengfang Jin et al., ALDH2(E487K) mutation increases protein turnover and (ECL) promotes murine hepatocarcinogenesis. Proc Natl Acad Sci U S A. 2015 Jul 21; Mutation was also found in human patient sample: DDTKTLIKTIVTRIND 112(29): 9088–9093. (A) MDA-MB-231 cells were transiently transfected with Cas9-gRNA #3 targeting gene X. Clones were picked and grown up for WB analysis for Gene X expression. (B) 2-D colony formation assays for MDA-MB-231 clones with gene X deleted. onco.wuxiapptec.com Email: [email protected] Oncology & Immunology Oncology CRISPR-Cas9 based genome-wide knockout screening Provide top level genome editing service for drug discovery • To understand drug resistance and target validation Target Clients Libraries design and construction Expansion of CRISPR lentiviral libraries Transduce cells with libraries • Global pharmaceutical company • University and Institute • Biotech and CRO company IVF/microinjection CRISPR/CAS9 Animal Models Business Development 2nd generation sequencing and data analysis Untreated/drug screening Antibiotics selection Service & IP Disease models Translational Medicine In vivo CRISPR screen in syngeneic model for sensitizing Epi1 suppresses in vivo tumor growth or blocking mutations to immunotherapy In Vivo CRISPR Animal Model and Services • From genome editing to disease models and efficacy study Compliance and Accreditations • AAALAC accredited facility • OECD – received a statement of GLP compliance from the Belgium regulatory authorities • Chinese SFDA – received certificate of GLP compliance • WuXi has conducted IND and NDA enabling studies for submission to the US FDA Deletion of Epi1 sensitizes tumor to PD-1/PD-L1 blockade Transgenic Services Conditional Knockout Services • Over-expression of target genes(stable or inducible) • CRISPR/CAS9 based knockout/knockin in mouse, rat Knockout Services • ES cell based CKO in mouse • CRISPR/CAS9 knockout in mouse, rat and larger animals Cryopreservation Services • Embryonic stem cell based knockout in mouse Genotyping and Phenotyping Services Knockin Services • CRISPR/CAS9 based knockin in mouse, rat Oncology & Immunology onco.wuxiapptec.com.
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