Eric Paul Bennett, MSc, Dr.med. Cellular Indel Profiles and Dynamics CRISPR/Cas9 delivery formats: Applying in vitro methodologies ex vivo

Transcription

1 Copenhagen Center for Glycomics/Department of Odontology Eric Paul Bennett, MSc, Dr.med. Cellular Indel Profiles and Dynamics CRISPR/Cas9 delivery formats: Applying in vitro methodologies ex vivo

2 Eric Eric P. P. Bennett Bennett CCG/Department of Odontology Presentation Outline Glycomics meets genome editing: What s the link? grna design validation for all Cas9 applications Improved methods for enhanced targeting and indel profiling: FACS meets IDAA In vitro and ex vivo indel profiles and dynamics induced by different CRISPR/Cas delivery systems

3 50 ZFNs 5 TALENs Eric P. Bennett CCG/Department of Odontology Creating Isogenic Glycogene Engineered Cells Precise Gene Editing: 13 B.C. A.C. 200 CRISPRs validated! Hansen L., et al., Glycobiology, 2015, 25(2): Candy Floss, restaurant Geist Alberts, 2nd edition Nucleic Acid Res., 2017, 45(13):e123 Nature Protocols, 2017, 12(3): Nature Biotech. 2015, 33(8):842-4 Nucleic Acids Res. 2015, 19;43(9) Proc Natl Acad Sci U S A. 2014,111(39)3 Nucleic Acids Res. 2014, 42(10) EMBO J. 2013, 32(10): Proc Natl Acad Sci U S A. 2013, 110(52) Proc Natl Acad Sci U S A. 2012,109(25) Nat Methods. 2011, 8(11): ZFN ZFN/TALEN/CRISPR ZFN ZFN/TALEN/CRISPR ZFN ZFN/TALEN/CRISPR ZFN ZFN ZFN ZFN

4 Eric P. Bennett CCG/Department of Odontology CRIPSR/Cas9 and it s Repurposed Applications Hsu, Lander and Zhang, cell, 2014

5 Eric P. Bennett CCG/Department of Odontology The Underlying Principle for Cas9 Target Binding Knock out Knock in Nickase CRISPR-A CRISPR-I CRISPR-Modifier: Epigenetic Base Edit CRISPR applications: Garneau et al, 2010, Nature Haurwitz et al., 2010, Science Deltcheva et al, 2011, Nature Jinek et al., Science Cong et al, 2013, Science Mali et al., 2013, Science Gilbert et al, 2014, Cell Hilton et al., 2015, Nat Biotechnol Komor et al., 2016, Nature

6 Odontologisk Institut Factors affecting Cas9 targeting efficiency U6 promoter, guide RNA, trcrna/scaffold: TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTG CATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTT GGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAA GGACGAAACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA M. Kosicki, et al., Prog Mol Biol Transl Sci. 2017;152: doi: /bs.pmbts Epub 2017 Nov 6.

7 Cas9 targeting: The Chimeric grna design 2 component grna delivery most effective Cong et al., Science 2013

8 Factors Determining the grna Design Funcionality? Score L.Lonowski et al., Nat Protoc, 2017 Y. Narimatsu et al., manuscript accepted Gene name (HGNC) GALNT10 GALNT11 GALNT12 GALNT13 GALNT14 GALNT15 GALNT16 GALNT17 GALNT18 GALNT19 GALNT20 Protocol grna Sequence Validation* +=10-25%, ++=26-50%, +++= >50% Amplicon Major FACS Pool indel Bulk BbsI/QCG ACTCTCTCAGCATCGGTCAT BbsI/QCG CTCTCTCAGCATCGGTCATG QCgRNA amplicon ATCTGGGAGAGAGCGATTCA BbsI/QCG ATCGCTCTCTCCCAGATATC BbsI/QCG ATGCTTATCAGTGACCGCTT BbsI/QCG TATGCTTATCAGTGACCGCT QCgRNA amplicon CGATCAAGAGTTGAGAGACT /-2 BbsI/QCG CATCTCTGTGGTAGCCCAAG QCgRNA amplicon GGACACTGTAAACTGTCCGA BbsI/QCG TTCTTCTAGCAGGATATCCG BbsI/QCG GGATATCCGGGGATGTCTCA BbsI/QCG TGTCCGAAGGAGAGTTGACC QCgRNA amplicon TTAATACGTGCCCGTCTTCG QCgRNA amplicon ACTGTGAATGCACGTTAGGA QCgRNA amplicon TGAATGCACGTTAGGATGGC QCgRNA amplicon AAGCAGCTGCTCCTCGAAGA QCgRNA amplicon AGGTTAATGATATCGATCAC QCgRNA amplicon CTCCGAGGCAGACTCGATGT QCgRNA amplicon CACCTACATCGAGTCTGCCT QCgRNA amplicon GGCAGACTCGATGTAGGTGA QCgRNA amplicon TTGTCTTACAGGACTACACG QCgRNA amplicon CTTACAGGACTACACGCGGG QCgRNA amplicon GGTTAATGATATCGATCACA QCgRNA amplicon GGATGCTGTGTACAGTCCGC QCgRNA amplicon GCTGGCTGAGGTCGTCCACG QCgRNA amplicon CCTGAAGGAGATCATCCTCG QCgRNA amplicon TGATCCGGTCCCGAGTGCGT QCgRNA amplicon CCGCCCCACGCACTCGGGAC QCgRNA amplicon ACTGCGAAGTGAACACCGAG QCgRNA amplicon ACTCGGTGTTCACTTCGCAG QCgRNA amplicon TGATGAGAAGGCCTACCTGT /+1 QCgRNA amplicon TCAGCTTGTCACTCTCCAGC QCgRNA amplicon GTAATGGCGGGTGTCCCGGA QCgRNA amplicon CATCCGGACCCGTCTCCTGG /+1 QCgRNA amplicon AGTTCACATTGACCTCGCAG QCgRNA amplicon ATTCCTGGACTCCCACTGCG QCgRNA amplicon ATGGCGAGATGATCCGCTTC BbsI/QCG TGAGATAGAAGAGTACCCGC /+1 BbsI/QCG ACCTGTACTCACCCGCATCA BbsI/QCG TCCTTGATGCGGGTGAGTAC QCgRNA amplicon AGGACAACTTTGAGGTGCAG QCgRNA amplicon CAGCTCCCAGCTGTACCCGT QCgRNA amplicon CGTCCCACCAGTCTTTTGGG QCgRNA amplicon GTTGGTGTTGAACTTGATCG QCgRNA amplicon CGGCCCATCGCGGTGCGCAG QCgRNA amplicon AGGTTGGCCACCTCGGCGCG QCgRNA amplicon ATACTCTGTTCACCTCACAG QCgRNA amplicon CATCAATGACATCTATCAGG QCgRNA amplicon AGTTCCCCTTACAAGAGGAG /8 4/7 3/6 2/3

9 High Throughput grna Design Validation of 200 Glyco-Genes A L.Lonowski et al., Nat Protoc, 2017 Y. Narimatsu et al., manuscript accepted

10 GlycoCRISPR: Matrix of 200 Human validated Glycogene grna Designs with >30% cutting efficiency Copenhagen Center for Glycomics +1 as predominant indel event in >60% grna designs L.Lonowski et al., Nat Protoc, 2017 Y. Narimatsu et al., manuscript accepted D.Paquet et al., Nature, 2016 M. van Overbeek et al., Mol. Cell, 2016

11 Indel efficiency Copenhagen Center for Glycomics Indel Detection,,,, why and when? CRISPR/ Cas9 Cell pool Cellular DNA extraction? Indel Detection Insight Days 100%??? days post delivery indel profile/dynamics

12 The most Commonly used Indel Detection Methods NGS deep sequencing or Sanger EMC (Enzyme Mismatch Cleavage) T7EI/Cel-I/Surveyor Clone#2 Cell Pool ? Defined identification of individual indel events in cells Day K562, CRISPR/Cas9, tri-allelic KRAS targeting Undefined identification of indel events in cells 0 1 Days

13 ɸX-HaeIII Hela- D4E DNA control Main Enzyme Mismatch Cleavage Assay Issue T7EI EMC 13 * Y. Zhang et al., NAR, 2015 MC. Huang et al.,. Electrophoresis 33, 2012 L. Vouillot et al., G3 5, , 2015 T. Sakurai, et al., BMC Biotechnol., bp Possess background nonspecific activity and exonucleolytic avtivity T7EI has preference minor for indels heteroduplex DNA formed by deletions wt Insufficient detection of

14 RFU Indel Detection by Sequenator based Fragment Analysis Indel Detection by Amplicon Analysis (IDAA) A Precise Gene Targeting 14 Zhang Yang DSB Single base size discrimmination power of the Sequenator! NHEJ deletion insertion B * Tri-primer PCR C * * * deletions 0.2% wt 10% 11% 6% 0.5% 6% % 0/wt deletions insertions insertions amplicon size/bp indel size/bp Detection Amplicon electrohoresis Z. Yang et al., Nucleic Acids Res. 2015

15 RFU * Copenhagen Center for Glycomics IDAA for Genome Targeting Indel Profiling Tri-primer PCR HeLa COSMC-ZFN Targeting 872 ɸX -1 WT -1bp/Hela- D4E IDAA WT Bp * * * WT IDAA TM T7E/ EMC 345 K562 CRISPR/Cas9, triallelic KRAS Targeting Clone#2 Pool Clone#2 IDAA WT Cell pool Z. Yang et al., Nucleic Acids Res T7E/ EMC

16 IDAA Indel Profile & Detection Sensitivity similar to NGS CHO mono allelic C1galt1c1 (Cosmc) locus) grna2 : GAATATGTGAGTGTGGATGGAGG d0 d2 + grna2 Cell Pool DNA lysate 1X10 6 cells NGS/MiSeq indel profile vs IDAA indel profile 6-FAM + F IDAA R Indel Detection by Amplicon Analysis Slide 16

17 Fragments/reads RFU Fragments/reads IDAA Indel Profile & Detection Sensitivity similar to NGS ABI3500xl 285 Zoom in IDAA amplicon size/bp indel size/bp 345 amplicon size/bp Reliable Sanger detection limit 17% S. Jamuar et al., N ENGL J MED, Reliable T7EI (EMC) detection limit 2-5% Y. Fu, J. Sander, Nat. Biotech, 2013 indel size/bp NGS detection limit as low as 0.12% 0,01 S. Jamuar et al., N ENGL J MED; 2014, Tsai, Nat Biotech, 2015 % relative to wt peaklog scale Zoom in 26,5% 11,5% 19,6% 10,5% Peak number NGS Indel Detection Sensitivities ,1 % relative to wt unmodified peak=0 IDAA%/wt MiSeq% 0,59% 0,22% 0,31% 0,23% 0,13% 0,09% % L.Lonowski, Nat Protoc, 2017

18 Copenhagen Center for Glycomics IDAA Indel Finger Print ; grna Design Specific K562 HEK293 L.Lonowski, Nat Protoc, 2017

19 23/11/ Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System Zetsche et al., Cell (2015), Recognizes a T-rich protospacer-adjacent motif (PAM) Cpf1 creates sticky ends (4-5 nt) Cpf1 Cas9 S. Stella, P. Alcón, G. Montoya, Nat Struct Mol Biol

20 Acidaminococcus (Ac)Cpf1 PAM Specificity 23/11/ T.Yamano et al., Mol Cell, 2017 S.Stella et al., Nature, 2017

21 Mark Behlke AsCpf1 IDAA Indel Fingerprint 23/11/ WT Vice President, IDT Cpf1 nucleus Cpf1 grna#1-5 TTTA HEK293-Cpf1 Cpf1 grna#2 TTTC Cpf1 sgrna Cpf1 grna#3-6 TTTC Cpf1 grna#4 TTTC Unpublished

22 23/11/ ZFN and TALEN IDAA Indel Finger Print HEK L.Lonowski, Nat Protoc, 2017

23 Methods for Indel Profiling after Genome Editing M. Kosicki, et al., Elsevier, 26 th November, 2017

24 Sanger Institute Copenhagen Center for Glycomics Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods Metzakopian Emmanouil M. Kosicki, et al., Prog Mol Biol Transl Sci. 2017;152: doi: /bs.pmbts Epub 2017 Nov 6.

25 Eric P. Bennett CCG/Department of Odontology Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods Human ST6GALNACI +1

26 Morten Frödin Genome Editing Workflow based on FACS and IDAA A BRIC, KU + grna B L.Lonowski et al., Nat Protoc, 2017 Z.Yang et al., Nat Biotech, 2015

27 Copenhagen Center for Glycomics Dynamics of Indel Profiles Induced by Cas9-sygRNA (RNP) Delivery WT ST6GALNACI WT M. Kosicki, et al., Prog Mol Biol Transl Sci. 2017;152: doi: /bs.pmbts Epub 2017 Nov 6.

28 Copenhagen Center for Glycomics Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods M. Kosicki, et al., Prog Mol Biol Transl Sci. 2017;152: doi: /bs.pmbts Epub 2017 Nov 6.

29 Eric Eric P. P. Bennett Bennett CCG/Department of Odontology In vitro and ex vivo Indel Profiles and Dynamics Develop in uniformly and proportionally over time Depend on the CRISPR/Cas delivery systems used Depend on the repair capacity of the cells targeted Highly reproducible for a given grna design used in a given cell( line) ( finger print profile )

30 IDAA Reagents and Services Ship Amplicons 30