The CRISPR-Cpf1 nuclease is the best alternative to the commonly used Cas9 for genome editing. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence of TTTV, which differs from the Cas9 PAM sequence, NGG. Having Cpf1 as a second option increases the likelihood of editing as close as possible to your desired target site. In this presentation, Dr Rolf Turk introduces the optimized Alt-R™ CRISPR-Cpf1 System and explains how it can be used as a powerful new tool for your genome editing research. Dr Turk presents the basics of the system, as well as protocols for getting started in genome editing.

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

3. 3

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
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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%
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13. Alt-R™ CRISPR-Cpf1 RNP complex
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14. Alt-R™ CRISPR-Cpf1 crRNA—protospacer length optimization
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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
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Locations affected by 2′OMe modification
T7EI total editing (%) T7EI total editing (%)

16. 16

17. Alt-R™ CRISPR-Cpf1 RNP complex formation
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18. Effect of Alt-R™ Cpf1 Electroporation Enhancer with RNP
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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
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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
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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
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0
10
20
30
40
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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
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* 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.
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31. THANK YOU
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32. Questions?
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