Cpf1-based genome editing using ribonucleoprotein complexes
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The document discusses the optimization and applications of the alt-RTM CRISPR-Cpf1 genome editing system, covering various aspects such as crRNA optimization, delivery methods as RNP complexes, and homology-directed repair in mammalian cells. It compares the efficiency and specificity of Cpf1 to Cas9 and highlights the advantages of Cpf1, including lower off-target effects and dependency on PAM site sequences. The document also provides data on various experiments conducted to evaluate editing efficiency and methods for enhancing delivery through electroporation.
Cpf1-based genome editing using ribonucleoprotein complexes
- 1. Cpf1-based genome editing using the Alt-R™ CRISPR-Cpf1 System Rolf Turk, PhD 1
- 2. Outline: Alt-R™ CRISPR-Cpf1 System • Background • Optimization of Cpf1 crRNA • Length optimization • Chemical modification • Delivery of Cpf1 as ribonucleoprotein (RNP) complex • Effect of Alt-R Cpf1 Electroporation Enhancer • RNP concentration optimization • Homology-directed repair using Cpf1 • Positive controls 2
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- 4. Cas9 genome editing • RNA-guided endonuclease • PAM site (NGG) • crRNA and tracrRNA • Blunt-ended cut sites 4
- 5. Cpf1 genome editing • RNA-guided endonuclease • Cpf1: CRISPR from Prevotella and Francisella 1 • Class II, type V • Cpf1 editing in mammalian cells • Acidaminococcus sp. BV3L6 • Lachnospiraceae bacterium ND2006 • Single guide RNA (crRNA, 41–44 nt) • Double-stranded break with staggered ends • PAM site is thymidine-rich • Preferentially uses TTTV 5 Zetsche B, Gootenberg JS, et al. (2015) Cpf1 is a single RNA-guided Endonuclease of a class 2 CRISPR-Cas system. Cell, 163:759–771.
- 6. Cpf1 structure and function 66 Yamano T, Nishimasu H, et al. (2016) Crystal structure of cpf1 in complex with guide RNA and target DNA. Cell, 165(4):949–962.
- 7. Comparison of Cas9 and Cpf1 7
- 8. Low off-target editing with Cpf1 8 Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.
- 9. Lower off-target effects with Cpf1 RNP vs. plasmid 9 Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.
- 10. Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency 10 0 10 20 30 40 50 60 70 80 90 100 46550 46650 46750 46850 46950 47050 47150 47250 T7EItotaleditingefficiency(%) Chromosome location STAT3: exons 5 and 6 HEK 293—RNP—Amaxa® Nucleofector® System AsCpf1 SpCas9 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 T7EItotaleditingefficiency(%) Ranked editing efficiency STAT3: exons 5 and 6 HEK 293—RNP—Amaxa® Nucleofector® System AsCpf1 SpCas9 93% 35% 15%
- 11. A.s. Cpf1 editing efficiency is PAM-sequence dependent 11 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 T7EItotaleditingefficiency(%) HEK 293—RNP—Amaxa® Nucleofector® System 232 crRNAs across 6 genes TTTA TTTC TTTG TTTT Ranked editing efficiency
- 12. Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 T7EItotaleditingefficiency(%) Ranked editing efficiency STAT3: exons 5 and 6 HEK 293—RNP—Amaxa® Nucleofector® System AsCpf1 (TTTN) AsCpf1 (TTTV) SpCas9 (NGG) 93% 35% 15% 53% 12
- 13. Alt-R™ CRISPR-Cpf1 RNP complex 13
- 14. Alt-R™ CRISPR-Cpf1 crRNA—protospacer length optimization 14 0 10 20 30 40 50 60 70 80 90 100 38171-AS 38254-AS 38325-S 38337-AS 38351-S 38538-S T7EItotaleditingefficiency(%) HPRT1 crRNA location and guide strand HEK 293–Cpf1 Stable Cell Line—30 nM crRNA RNAiMAX™ (Thermo Fisher) 24 mer 23 mer 22 mer 21 mer 20 mer
- 15. Alt-R™ CRISPR-Cpf1 crRNA—2′O-methyl testing 15 Locations affected by 2′OMe modification T7EI total editing (%) T7EI total editing (%)
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- 17. Alt-R™ CRISPR-Cpf1 RNP complex formation 17
- 18. Effect of Alt-R™ Cpf1 Electroporation Enhancer with RNP 18 0 10 20 30 40 50 60 70 80 90 100 T7EItotaleditingefficiency(%) HPRT1 crRNA location and guide strand HEK 293—5 µM RNP—Amaxa® Nucleofector® System 0 µM Enhancer 3 µM Enhancer 5 µM Enhancer
- 19. Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector® 1919 * * = Toxicity 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 T7EItotaleditingefficiency(%) Cpf1 ribonucleoprotein complex concentration (µM) HEK 293—RNP—Amaxa® Nucleofector® System HPRT1 38228-S No Enhancer Equimolar Enhancer 3 µM Enhancer
- 20. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 T7EItotaleditingefficiency(%) Cpf1 ribonucleoprotein complex concentration (µM) HEK 293—RNP—Amaxa® Nucleofector® System HPRT1 38330-AS No Enhancer Equimolar Enhancer 3 µM Enhancer 20 * * = Toxicity Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector®
- 21. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 T7EItotaleditingefficiency(%) Cpf1 ribonucleoprotein complex concentration (µM) HEK 293—RNP—Neon® Transfection System HPRT1 38330-AS No Enhancer Equimolar Enhancer 1.8 µM Enhancer Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Neon® 21 ** * = Toxicity
- 22. Alt-R™ CRISPR-Cpf1 RNP editing efficiency—Nucleofector® vs. Neon® electroporation 22 0 10 20 30 40 50 60 70 80 90 100 38115-AS 38186-S 38228-S 38330-AS T7EItotaleditingefficiency(%) HPRT1 crRNA location and guide strand HEK 293 Cells NFXN Neon 5 µM RNP 3 µM Enhancer 5 µM RNP 1.8 µM Enhancer
- 23. Alt-R™ Cpf1 Electroporation Enhancer does not alter indel profile 23 Indel Size Distribution - NGS - HEK293 Indel size (bp) -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 Sequences(%) 0 5 60 80 100 0 µM Cpf1 Electroporation Enhancer 3 µM Cpf1 Electroporation Enhancer
- 24. Alt-R™ CRISPR-Cpf1 editing—time course 24 0 10 20 30 40 50 60 70 80 90 100 15 25 35 45 55 65 75 T7EItotaleditingefficiency(%) Time post-transfection (hr) 0 10 20 30 40 50 60 70 80 90 100 15 25 35 45 55 65 75 Time post-transfection (hr) 38115-AS 38186-S 38228-S 38330-AS HEK293 HeLa 5 µM RNP—3 µM Enhancer—Amaxa® Nucleofector ® System T7EItotaleditingefficiency(%)
- 25. 25 Flanking arm length 92 72 57 47 37 27 Cpf1 cleavage crRNA guide GAATTC (EcoRI) Total length ssODN 190 150 120 100 80 60 Homology-directed repair in HEK 293–Cpf1 stable cell line
- 26. Homology-directed repair in HEK 293–Cpf1 stable cell line 26 0 10 20 30 40 50 60 70 80 90 100 190 150 120 100 80 60 190 150 120 100 80 60 Non-targeted strand (nt) Targeted strand (nt) T7EItotaleditingefficiency(%) HEK 293—HPRT1 38343-S RNAiMAX®—30 nM crRNA—30 ng HDR template (ssODN) EcoRI T7
- 27. Alt-R™ CRISPR-Cpf1 RNP delivery using lipofection 27 0 10 20 30 40 50 60 70 80 90 100 1:1 2:1 5:1 1:1 2:1 5:1 1:1 2:1 5:1 10 nM Cpf1 30 nM Cpf1 50 nM Cpf1 T7EItotaleditingefficiency(%) Cpf1 concentration and ratio crRNA:Cpf1 HEK 293—RNAiMAX™ * =Toxicity * * * * *
- 28. Positive controls for genome editing using the Alt-R™ CRISPR-Cpf1 System 28 0 10 20 30 40 50 60 70 80 90 100 Hs - HEK 293 Mm - Hepa1-6 Rn - RAT2 T7EItotaleditingefficiency(%) 5 µM RNP—3 µM Enhancer Amaxa® Nucleofector® System
- 29. Comparison of genome editing using SpCas9 vs. AsCpf1 29 * Molecular weight of Alt-R™ Nuclease † N = any base V = A, C, or GTTTV
- 30. Alt-R™ CRISPR-Cpf1 System Core components: • Alt-R CRISPR-Cpf1 crRNA • Alt-R A.s. Cpf1 Nuclease 2 NLS • Alt-R Cpf1 Electroporation Enhancer Sequences for positive and negative crRNA controls for human, mouse, and rat are available at www.idtdna.com/CRISPR-Cpf1. Also see our highly effective Alt-R CRISPR-Cas9 System at www.idtdna.com/CRISPR-Cas9. 30
- 31. THANK YOU 31
- 32. Questions? 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 “Best tech support ever, @idtdna!” Lauren Sakowski 32