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Making the cut with CRISPR

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A quick introduction to the technical challenges facing biologists when editing genomes using CRISPR/cas9 technology.

Published in: Health & Medicine
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Making the cut with CRISPR

  1. 1. MAKING THE CUT WITH CRISPR TECHNICAL CONSIDERATIONS FOR EDITING THE GENOME -SXSW 2016-
  2. 2. WHY SHOULD CRISPR LOOK LIKE THIS?
  3. 3. WHEN IT COULD LOOK LIKE THIS?
  4. 4. AGENDA July 15 4 1. Brief intro to Desktop Genetics 2. CRISPR genome editing overview 3. Considerations in CRISPR design 4. Hand-on Demo 5. Q&A
  5. 5. DESKTOP GENETICS COMPANY SNAPSHOT London-based software company founded 2012: Enabling “ lit eral D eskt op Genet ics” Giving biologist s t he t ools t o edit any gene w ith ease Our expertise: Vis ua lis a t ion | U X D esign D N A Search | D N A A ssembly | Genome Edit ing July 15 5
  6. 6. DESKGEN PLATFORM DESIGN ANY GENOME EDITING EXPERIMENT FROM YOUR DESKTOP July 15 6 FREE SOFTWARE FOR ACADEMICS COMMERCIAL SUBSCRIPTIONS ADVANCED GENOMIC SERVICES
  7. 7. WHO WORKS WITH US PARTNERS AND COLLABORATORS BENEFITING FROM THE PLATFORM Microorganism engineering Cambridge, MA • DNA search engine and automated cloning algorithms Genome editing company, Cambridge UK • Internal cell line engineering tool • Academic-facing “gUIDEbook” CRISPR therapeutic company, Cambridge MA • Design and assess the specificity of CRISPR-based therapeutics Cancer research center Madrid, Spain • Design libraries for cancer pathway mapping July 15 7
  8. 8. CRISPR IS A BACTERIAL IMMUNE SYSTEM July 15 WE’VE LEARNED HOW TO HIGHJACK IT 8
  9. 9. ANATOMY OF A CUT July 15 S. PYOGENES CAS9 CUTS GENOME UPSTREAM OF “NGG” MOTIF 9 Two cutting domains: • HNH • RuVC Constant scaffold for Cas9 binding Jinek et al., Science 2012 Hsu et al., Nature 2013
  10. 10. July 15 10 GENOME EDITING MECHANISMS USING THE CELL’S REPAIR PATHWAYS TO ENGINEER THE GENOME HIGH FREQUENCY ER R OR - PR ON E LOW FREQUENCY H IGH FID ELITY Non Homologous End Joining (NHEJ) Homology Directed Repair (HDR) Double Stranded Break
  11. 11. July 15 11 GENOME EDITING TECHNIQUES CRISPR IS A RAPID AND EFFECTIVE GENOME ENGINEERING METHOD Zinc Finger Nuclease TAL Effector Nuclease CRISPR Programmable Protein Protein DNA Engineering Complex Complex Easy Specificity Med High Med/High Multiplex No No Yes Species Few Few Many
  12. 12. July 15 12 ACCESSIBLE AND WIDESPREAD RAPIDLY ADOPTED TECHNOLOGY – REQUESTS FROM ADDGENE Source: http://www.blog.addgene.org/trends-in-crispr-and-synbio-technologies-slideshare
  13. 13. July 15 13 APPLICATIONS OF CRISPR/CAS9 VERSATILE TOOL GOES BEYOND CUTTING DNA Mali et al., Nature 2013
  14. 14. CONSIDERATIONS OF EXPERIMENTS July 15 14 IT ALL STARTS WITH THE DESIGN OF GUIDE RNAS  Experimental intent  Accurate data models  Off-target activity  On-target activity  Delivery technique
  15. 15. EXPERIMENTAL INTENT July 15 15 WHAT DO I WANT TO DO? Experimental intent determines genomic location: – KNOCK-OUT  prefer 5’ targeting – KNOCK-IN  cut within ~30 bp of foci – ACTIVATE  target within 200bp 5’ of TSS – INHIBIT  target ± 200bp around TSS Always consider all transcripts and coding / non-coding regions GENOMIC CONTEXT MATTERS
  16. 16. July 15 16 DESIGNING GUIDES THE MORE YOU KNOW ABOUT YOUR TARGET, THE BETTER Shi et al., Nature Biotechnology 2015 GENOMIC CONTEXT MATTERS
  17. 17. REFERENCE VS ACTUAL GENOME July 15 17 SNPs can result in widely different gRNA activity Reference -> Real Genome Sequence SNP location Activity score G -> A PAM site 0.69 -> 0.00 - 1 0 0 % AC T I V I T Y D I F F E R E N C E G > A TP53 chr17: bp 7676532 rs1800369
  18. 18. REFERENCE VS ACTUAL GENOME July 15 18 SNPs can result in widely different gRNA activity G > A PLK1 + 5 X AC T I V I T Y D I F F E R E N C E Reference -> Real Genome Sequence SNP location Activity score G -> A Seed region 0.01 -> 0.05 chr16: bp 23680098 rs547328721
  19. 19. REFERENCE VS ACTUAL GENOME July 15 19 CONCLUSION “USE THE ACTUAL GENOME OF YOUR CELL LINE AND NOT THE REFERENCE GENOME” - George Church - personalised genome editing
  20. 20. CRISPR SPECIFICITY July 15 20 WHERE ELSE MIGHT MY GUIDE CUT? Cas9 is tolerant of RNA-DNA mismatches (up to 6 shown) Score range: 0 (low specificity) 100 (high specificity) Important: Consider locus of off-target Scan entire genome Hsu et al., Nature 2013
  21. 21. July 15 21 CRISPR SPECIFICITY IT’S NOT “IF” BUT “WHERE” THAT MATTERS DO I CARE? How “RISKY” is this guide? WHERE else does this cut? Do I care? Example output of DESKGEN off-target analysis: 1 2
  22. 22. INCREASING SPECIFICITY July 15 22 NICKASE PAIRS WITH D10A REDUCED OFF-TARGET REDUCED EFFICIENCY
  23. 23. ON TARGET ACTIVITY July 15 23 HOW WELL WILL MY GUIDE CUT? Activity score indicates probability a cut will occur Score range: 0 (low activity) 100 (high activity) Derived from machine- learning analysis trained on 1,841 guides Doench et al., Nature 2014 NOT ALL GUIDES ARE CREATED EQUAL
  24. 24. July 15 24 ON TARGET ACTIVITY NOT ALL GUIDES ARE CREATED EQUAL Source: internal project, Desktop Genetics
  25. 25. DELIVERY TECHNIQUES July 15 25 DELIVERING DNA • PLASMID – Single construct – Higher efficiency – Extended time to cutting & increased toxicity – More events: on-target and more off-target • PCR AMPLICON + CAS9 – Co-transfect PCR amplicon with Cas9 plasmid – Higher throughput – Two construct system
  26. 26. DELIVERY TECHNIQUES July 15 26 OTHER DELIVERY MECHANISMS PRE-TRANSCRIBED mRNA – Co-transfect RNA of Cas9 & gRNA /scaffold – Reduced toxicity, faster effect, more transient effect CAS9 PROTEIN COMPLEXED WITH gRNA – Rapid cutting activity observed – Reduced delivery into cell, reduced on-target cutting VIRAL DELIVERY – Typical approach for targeting cells in vivo – Lentiviral packaging – Bioproduction element – Payload may be too large with S. pyogenes – Smaller Cas9 orthologues required
  27. 27. July 15 27 HANDS-ON DEMO WWW.DESKGEN.COM
  28. 28. July 15 28 COLLABORATE & LEARN WWW.DESKGEN.COM
  29. 29. CUSTOM LIBRARIES ONE STOP SHOP FOR CUSTOM CELL LINE SPECIFIC LIBRARIES • You own library – design  data • Pooled or arrayed Additionally: • TET inducible promoters • sgRNA-Cas9 lentiviral vector containing fluorescence reporters July 15 29
  30. 30. EDWARDP@DESKGEN.COM W W W . D E S K G E N . C O M
  31. 31. July 15 31 GUIDE DETAILS EXPLANATION OF GUIDE RNA INFORMATION OFF-TARGET SCORE Hsu et al., 2013 How the score is calculated: 1. Start at 100 (very specific) 2. Subtract all off-target sites scores 0 (low specificity) 100 (high specificity) ACTIVITY SCORE Doench et al., 2014 0 (low activity) 100 (high activity) GC% Stay within 20-80%
  32. 32. July 15 32 DATA READOUT: SURVEYOR ASSAY FRAGMENT C = 650bp (size of PCR product) FRAGMENT B = 370bp FRAGMENT A = 280bp 500-800bp GENOMIC PCR CLEAN UP MELT & RE- ANNEAL DIGEST with SURVEYOR nuclease RUN on a GEL QUANTIFY
  33. 33. July 15 33 DESKGEN PLATFORM IS UNIQUE COMPREHENSIVE AND PERSONALISED CRISPR DESIGN Off-target Activity Knock-In Genomic data Vector construct. Nickase pairs # Genomes DESKGEN X X X X X X ANY Chopchop X * 10+ E-crisp X * X 10+ Crispr- design X * X 10+ Cosmid X * 2 Benchling X * X X X 10 Sgrna Designer X 2 Crispr-era X* X 2 * = Heurstics based, NOT comprehensive or exhaustive search

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