Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products

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2 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products which one is right for you? CRISPR Workflow abm s Toolbox and Services Website Navigation ABM s Advantage

3 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products which one is right for you? CRISPR Workflow- abm s Toolbox and Services Website Navigation ABM s Advantage

4 The CRISPR-Cas9 Time Line Found in additional bacteria, archae Similarity of some CRISPR element to viral DNA Successfully used in model organisms Zebrafish, C. elegans, Rat, Rice, Human present (first ever description of repeated Sequences in E.coli: function unknown Renamed as CRISPR and associated proteins cas 1-4 described Bioinformatics tools developed, Various structural studies Genome editing possibilities using CRISPR-Cas system

5 (1) Research - Gene Knock out, knock in - Random mutations - Over expression, RNAi (2) Industry - Making a better organism for crops, livestock, biofuel, bioproduction (3) Medicine - Fixing genetic defects - Altering physiology The power of Genome Editing

6 Cas9 Cas9

7 Zn-finger / TALEN Cas9 Zn-finger / TALEN Zn-finger / TALEN Zn-finger / TALEN New sequence to design = 20 nt Nuclease itself UNALTERED New sequence to design = >1000 nt Nuclease itself an extra ~340 aa

8 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products which one is right for you? CRISPR Workflow- abm s Toolbox and Services Website Navigation ABM s Advantage

9 Cleavage must take place next to a Proteospacer- Adjacent Motif (PAM) For the most commonly-used Cas9 NGG Cas9 introduces a double-strand break The break is repaired by Non-Homologous End Joining (NHEJ) This introduces an Insertion or Deletion (InDel) resulting in a frameshift

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11 Using Cas9 nickase boosts specificity The downside = more challenging experiment design

12 CRISPR Knock out -GFP

13 dcas9 can recruit proteins of interest to specific genomic locations

14 Gene Activation in HEK293T cells using SAM. RNA expression analysis of 2 individual genes, RHOXF2(A) and MIAT(B) in HEK293T cells transfected with pooled sgrna s and dcas9-sam construct, calculated relative to the WT samples. Samples transfected with pooled sgrna s alone were used as a negative control. GAPDH was used as a housekeeping control.

15 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products which one is right for you? CRISPR Workflow- abm s Toolbox and Services Website Navigation ABM s Advantage

16 Lentiviral vector Lentivirus Adenovirus Integrating (permanent) Non-integrating (transient)

17 Constitutive Inducible REQUIRED!

18 Two Vector System All-in-One Vector System

19 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 Library Products which one is right for you? CRISPR Workflow- abm s Toolbox and Services Website Navigation ABM s Advantage

20 YES Nuclease or Nickase Nuclease Permanent Permanent or transient Transient EF1α-Cas9 + sgrna Cas9-sgRNA all-in-one Cumate-inducible Cas9 Requires CymR! Cas9 adenovirus sgrna - separate Lentivectors Lentiviruses Nickase Two vector system Two sgrnas per cleavage No induction Lentivectors Lentiviruses KO/KI NO dcas9 cloning vector Custom cloning service dcas9-sam Transcription activation dcas9-gfp Genomic locus visualizing

21 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 Library Products which one is right for you? CRISPR Workflow- abm s Toolbox and Services Website Navigation ABM s Advantage

22 Identify Target Choose a system Work with your cell line (in vitro) Verify your work Search our library products! Lentivirus, Adenovirus, Protein, AAV (soon) Establish your Stable KO cell line Surveyor Assay Sequencing Cas9 Expressing Cells Genome-wide KO Cell Lines *Verified via Sequencing Cleavage Detection Kit (Cat# G932) CRISPR-Cas9 Targeted Amplicon-Seq (Cat# IA00100)

23 Cas9 Expressing Stable Cell Lines CRISPR Genome wide KO Cell Lines CRISPR Genomic Cleavage Detection Kit

24 This is completed using targeted amplicon sequencing of the Cas9 Target Site to quantify the amount of knockout allele expressed in the cell sample versus the wild-type. In this particular experiment (first image from the left), we see complete knockout of mouse LIF gene (accession NM_008501) in C26 cells using our All-in-One Vector (abm cat# K ).

25 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 Library Products which one is right for you? Our CRISPR Toolbox and Services Website Navigation ABM s Advantage

26 Direct search of genes & filter options Orange quick link Promotional CRISPR Knock-Out Box

27 Direct search of genes & filter options Orange quick link Promotional CRISPR Knock-Out Box

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29 Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 Library Products which one is right for you? Our CRISPR Toolbox and Services Website Navigation ABM s Advantage

30 (1) Product Diversity (2) Technical Expertise (3) Customer Support (4) Economical Value (Affordable pricing with high quality)

31 WB image from Nature Paper using Pgam5 sgrna CRISPR/Cas9 All-in-one Lentivector (Mouse Target 1); Cat# K Kang, Y. J. et al. "Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling." Nat. Commun. (2015) 6:8371 doi: /ncomms9371 Jiang, G. et al. "Isorhapontigenin (ISO) inhibits invasive bladder cancer (BC) formation in vivo and human BC invasion in vitro by targeting STAT1/FOXO1 Axis." Cancer Prev Res. Published Online First April 14, doi: / CAPR Okugawa, Y. et al. "Clinical significance of SNORA42 as an oncogene and a prognostic biomarker in colorectal cancer." Gut (2015) doi: /gutjnl

32 "We are very happy with the product and the turn around time, and would therefore order another reagent." Dr. Alexandra Duverger, University of Alabama at Birmingham, CRISPR "The All-in-One Lentivectors worked very well." Dr. Makoto Miyazaki, University of Colorado-Denver, CRISPR

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34 Heidi Chu Department of Business Development Applied Biological Materials Inc Viking Way Richmond, British Columbia, Canada V6V 2J5 Office: 1-(604) ext 37 Skype: