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Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing efficiency Rolf Turk PhD, Staff Scientist Integrated DNA Technologies Outline—Alt-R™ CRISPR-Cas9 System • Ribonucleoprotein complex (RNP) – Generation of RNP complex – Stability of RNP complex – Editing efficiency of RNP • Cas9 electroporation enhancer – Increases editing efficiency • RNP delivery using the Amaxa® Nucleofector® System (Lonza) – Optimization • RNP delivery using the Neon® System (Thermo Fisher) – Optimization CRISPR genome editing CRISPR guide RNAs DNA incorporation or gene knockout • Homology-directed repair—add or replace gene sequences • Non-homologous end joining— destroy a gene Cas9 protein RNA-directed dsDNA endonuclease Implementing CRISPR-Cas9 gene editing CRISPR-Cas9 products from IDT 3-step transfection using Alt-R™ CRISPR-Cas9 System Step Step Step + + gRNA complex formation RNP complex formation RNP delivery 15 minutes 10–20 minutes 30–60 minutes Cas9 protein is rapidly degraded—fewer off-target effects Western blot Off-target indel production Liang X, Potter J, et al (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection J Biotechnol, 208:44–53 Benefits of RNP: Cas9 protein rapidly degraded, fewer OTEs RNP, HEK-293 cells Nucleofection, EMX1 targeting gRNA, 48h 50 45 40 35 30 25 20 15 10 T7EI cleavage (%) T7EI cleavage (%) HEK-293–Cas9 cells Nucleofection, EMX1 targeting gRNA, 48h 0.1 µM 0.3 µM On target µM µM Off target 50 45 40 35 30 25 20 15 10 0.1 µM 0.3 µM On target 1.0 µM 5.0 µM 10 µM Off target • Sustained expression of Cas9 allows for OTEs Even lowexpression HEK-293–Cas9 cells allow for off target editing • RNP is “fast on and fast off,” and has fewer OTEs Ribonucleoprotein ease of use—highly stable Cas9 system T7EI cleavage (%) 5 month RNP complex stability—RNP stored at [1 µM] HEK293 cells, 10 nM RNP (1.2 µL), RNAiMAX (1.2 µL), 48 hr 100 90 80 70 60 50 40 30 20 10 HPRT site Fresh Complex –80°C –20°C HPRT site 4°C Cas9 Buffer Fresh Complex OptiMEM –80°C –20°C 4°C PBS • Loss in activity seen at –20°C (likely due to freeze–thaws) • Premade complexes are stable at 4°C and –80°C with no loss in activity CRISPR workflow Lipofection Easier to transfect cells Design gRNAs Assemble RNP Electroporation Harder to transfect cells, primary cells Microinjection Mouse model generation and other research organisms Collect genomic DNA Analyze 10 T7EI total editing efficiency (%) Nucleofection—RNP dilution series—HEK-293 cells The effect of carrier DNA and sequence on editing efficiency Conditions: • • • • • • 100 90 80 70 60 50 40 30 20 10 • • • 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Ribonucleoprotein complex concentration (µM) 4 µM electroporation enhancer No electroporation enhancer 4.5 • • • Amaxa Nucleofection Nucleofection Solution SF Protocol 96-DS-150 x 105 cells/Nucleofection HPRT locus: 38285 crRNA:tracrRNA:Cas9 = 1.2:1.2:1 RNP concentrations, 0.125–4 µM Electroporation enhancer DNA, µM 48 hr incubation, following Nucleofection 3X genomic DNA dilution Digest with T7EI, U Fragment Analyzer™ (Advanced Analytical Technologies) 21 T7EI total editing efficiency (%) Nucleofection—RNP dilution series—Jurkat cells The effect of carrier DNA and sequence on editing efficiency Conditions: • • • • • • 100 90 80 70 60 50 40 30 20 10 • • • 0.5 1.5 2.5 3.5 Ribonucleoprotein complex concentration (µM) 4 µM electroporation enhancer No electroporation enhancer 4.5 • • • Amaxa Nucleofection Nucleofection Solution SE Protocol 96-CL-120 x 105 cells/Nucleofection HPRT locus: 38285 crRNA:tracrRNA:Cas9 = 1.2:1.2:1 RNP concentrations, 0.125–4 µM Electroporation enhancer DNA, µM 48 hr incubation, following Nucleofection 3X genomic DNA dilution Digest with T7EI, U Fragment Analyzer™ (Advanced Analytical Technologies) 22 T7EI total editing efficiency (%) Nucleofection—RNP dilution series—K562 cells The effect of carrier DNA and sequence on editing efficiency Conditions: • • • • • • 100 90 80 70 60 50 40 30 20 10 • • • 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Ribonucleoprotein complex concentration (µM) 4 µM electroporation enhancer No electroporation enhancer 4.5 • • • Amaxa Nucleofection Nucleofection Solution SF 96-FF-120 protocol x 105 cells/Nucleofection HPRT locus – 38285 crRNA:tracrRNA:Cas9 = 1.2:1.2:1 RNP concentrations, 0.125–4 µM Electroporation enhancer DNA, µM 48 hr incubation, following Nucleofection 3X genomic DNA dilution Digest with T7EI, U Fragment Analyzer™ (Advanced Analytical Technologies) 23 24 Workflow • • • • • • HEK-293 cells: x 105 cells per electroporation HPRT 38285 RNP—crRNA:tracrRNA:Cas9 = 1.8:1.8:1.5 µM 12 µL volume (10 µL electroporated) Electroporation enhancer, 1.8 µM 24 different protocols – Voltage – Pulse width – Pulse length • gDNA isolation 48 hr after electroporation • T7EI assay 25 HEK-293 electroporation optimization—Neon® System Test Prot_01 Prot_02 Prot_03 Prot_04 Prot_05 Prot_06 Prot_07 Prot_08 Prot_09 Prot_10 Prot_11 Prot_12 ü Prot_13 Prot_14 Prot_15 Prot_16 Prot_17 Prot_18 Prot_19 Prot_20 Prot_21 Prot_22 ü Prot_23 Prot_24 Editing Efficiency (%) - Enhancer + Enhancer 31.2 39.0 44.8 44.5 23.3 33.8 41.5 44.6 22.3 34.9 46.2 30.2 42.6 50.5 61.1 12.3 27.3 41.4 53.0 36.2 47.4 51.5 60.9 52.0 63.2 59.9 59.9 42.4 49.3 58.8 61.1 39.9 53.9 62.4 48.9 51.4 66.2 67.4 24.9 37.5 46.4 60.4 52.6 57.2 59.7 61.6 SD (%) - Enhancer + Enhancer 0.2 0.7 0.5 1.3 0.8 1.4 1.0 0.8 1.5 0.7 0.8 0.7 0.9 0.5 1.0 0.5 0.7 1.0 0.5 1.3 0.8 1.0 1.0 3.0 8.1 3.7 3.2 1.6 1.2 1.2 1.6 2.3 2.4 4.8 2.1 1.8 2.1 2.7 2.2 0.6 1.8 1.8 1.8 1.2 1.0 1.4 Cell Density - Enhancer + Enhancer 3 4 2 3 3 3 3 3 4 4 2 3 4 4 4 4 3 2 Voltage (V) 1400 1500 1600 1700 1100 1200 1300 1400 1000 1100 1200 1100 1200 1300 1400 850 950 1050 1150 1300 1400 1500 1600 Pulse Width (ms) 20 20 20 20 30 30 30 30 40 40 40 20 20 20 20 30 30 30 30 10 10 10 10 Number 1 1 1 1 1 1 2 2 2 2 3 3 low cell density high cell density low editing high editing 26 T7EI editing (%): Presence of electroporation enhancer Neon® electroporation optimization—HEK-293 Effect of electroporation enhancer 80 70 #12 60 #22 50 40 30 20 10 0 10 20 30 40 50 60 70 80 T7EI editing (%): Absence of electroporation enhancer 27 Workflow • • • • • • Jurkat cells: x 105 cells per electroporation HPRT 38285 RNP—crRNA:tracrRNA:Cas9 = 1.8:1.8:1.5 µM 12 µL volume (10 µL electroporated) Electroporation enhancer, 1.8 µM 24 different protocols – Voltage – Pulse width – Pulse length • gDNA isolation 72 hr after electroporation • T7EI assay 28 Jurkat electroporation optimization—Neon® System Test Prot_01 Prot_02 Prot_03 Prot_04 Prot_05 Prot_06 Prot_07 Prot_08 Prot_09 Prot_10 Prot_11 Prot_12 Prot_13 Prot_14 Prot_15 Prot_16 Prot_17 Prot_18 Prot_19 Prot_20 Prot_21 Prot_22 Prot_23 Prot_24 ü Editing Efficiency (%) - Enhancer + Enhancer 0.0 0.0 53.9 64.4 59.0 71.0 0.0 78.2 60.1 64.7 22.0 49.1 41.9 62.9 57.7 63.7 60.2 74.4 14.2 29.8 34.6 53.6 27.0 73.5 22.7 54.5 51.6 69.4 59.9 74.2 68.0 62.0 0.0 12.7 23.7 35.1 9.7 56.7 53.9 73.7 41.9 59.2 12.2 66.2 61.1 71.1 37.0 75.7 SD (%) - Enhancer + Enhancer 0.0 0.0 2.7 1.8 0.5 4.5 0.0 1.3 3.1 13.2 0.6 16.3 3.2 6.8 0.9 15.2 2.4 3.1 1.4 0.4 0.9 0.1 1.7 3.8 1.3 4.6 1.9 0.8 3.0 0.7 2.6 11.2 0.0 0.3 0.9 2.0 0.6 3.1 3.6 7.2 1.4 1.4 1.1 0.2 1.7 2.2 2.8 0.4 Cell Density - Enhancer + Enhancer 3 2 4 3 2 2 2 2 4 3 3 4 2 Pulse Voltage (V) Width (ms) 1400 20 1500 20 1600 20 1700 20 1100 30 1200 30 1300 30 1400 30 1000 40 1100 40 1200 40 1100 20 1200 20 1300 20 1400 20 850 30 950 30 1050 30 1150 30 1300 10 1400 10 1500 10 1600 10 Number 1 1 1 1 1 1 2 2 2 2 3 3 low cell density high cell density low editing high editing 29 T7EI editing (%): Presence of electroporation enhancer Neon® electroporation optimization—Jurkat Effect of electroporation enhancer 90 80 #24 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 T7EI editing (%): Absence of electroporation enhancer 30 Genome editing in mouse primary T-cells Collaboration with Jennifer Barr & Scott Lieberman, Dept of Pediatrics, University of Iowa 31 IDT collaborator protocols available on the web 32 T7EI total editing efficiency (%) Nucleofection—Cpf1 RNP (5 µM), HPRT12, HEK-293 Effect of electroporation enhancer on editing efficiency 100 90 80 70 60 50 40 30 20 10 Conditions: • • • • • • • • • • • • Amaxa Nucleofection Nucleofection Solution SF Protocol 96-DS-150 3.5 x 105 cells/Nucleofection HPRT locus crRNA:Cpf1 = 1.2:1 RNP concentration, µM Electroporation enhancer DNA, µM 48 hr incubation, following Nucleofection 3X genomic DNA dilution Digest with T7EI, U Fragment Analyzer™ (Advanced Analytical Technologies) 0 µM 3 µM 33 Questions? “Best tech support ever, @idtdna!” Lauren Sakowski TALK TO A PERSON Our experts are available for consultation “The people at @idtdna are awesome A+ for customer service.” Nikolai Braun Contact us by web chat, email, or phone Find local contact details at: www.idtdna.com 34 THANK YOU! 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