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Cpf1-based genome editing using the Alt-R™ CRISPR-Cpf1 System

Rolf Turk, PhD

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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

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Cas9 genome editing

• RNA-guided endonuclease• PAM site (NGG)• crRNA and tracrRNA• Blunt-ended cut sites

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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

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.

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Cpf1 structure and function

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Yamano T, Nishimasu H, et al. (2016) Crystal structure of cpf1 in complex with guide RNA and target DNA. Cell, 165(4):949–962.

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Comparison of Cas9 and Cpf1

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Low off-target editing with Cpf1

Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.

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Lower off-target effects with Cpf1 RNP vs. plasmid

Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.

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Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency

46550 46650 46750 46850 46950 47050 47150 472500

102030405060708090

100

STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System

AsCpf1 SpCas9

Chromosome location

T7EI

tota

l edi

ting

effici

ency

(%)

0 10 20 30 40 50 60 70 80 90 1000

102030405060708090

100

STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System

AsCpf1 SpCas9

Ranked editing efficiency

T7EI

tota

l edi

ting

effici

ency

(%)

93%

35%15%

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A.s. Cpf1 editing efficiency is PAM-sequence dependent

0 10 20 30 40 50 60 70 800102030405060708090

100

HEK 293—RNP—Amaxa® Nucleofector® System232 crRNAs across 6 genes

TTTATTTCTTTGTTTT

T7E

I tot

al e

ditin

g ef

ficie

ncy

(%)

Ranked editing efficiency

Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency

0 20 40 60 80 1000

10

20

30

40

50

60

70

80

90

100

STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System

AsCpf1 (TTTN) AsCpf1 (TTTV) SpCas9 (NGG)

Ranked editing efficiency

T7EI

tota

l edi

ting

efficie

ncy

(%)

93%

35%

15%

53%

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Alt-R™ CRISPR-Cpf1 RNP complex

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Alt-R™ CRISPR-Cpf1 crRNA—protospacer length optimization

38171-AS 38254-AS 38325-S 38337-AS 38351-S 38538-S0

102030405060708090

100

HEK 293–Cpf1 Stable Cell Line—30 nM crRNARNAiMAX™ (Thermo Fisher)

24 mer23 mer22 mer21 mer20 mer

HPRT1 crRNA location and guide strand

T7EI

tota

l edi

ting

effici

ency

(%)

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Alt-R™ CRISPR-Cpf1 crRNA—2′O-methyl testing

Locations affected by 2′OMe modification

T7EI total editing (%) T7EI total editing (%)

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Alt-R™ CRISPR-Cpf1 RNP complex formation

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Effect of Alt-R™ Cpf1 Electroporation Enhancer with RNP

38094-S

38104-S

38115-AS

38146-AS

38164-AS

38164-S

38186-S

38228-S

38330-AS

38343-S

38455-S

38486-S0

102030405060708090

100

HEK 293—5 µM RNP—Amaxa® Nucleofector® System

0 µM Enhancer3 µM Enhancer5 µM Enhancer

HPRT1 crRNA location and guide strand

T7EI

tota

l edi

ting

effici

ency

(%)

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Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector®

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*

* = Toxicity

0 1 2 3 4 5 60

10

20

30

40

50

60

70

80

90

100

HEK 293—RNP—Amaxa® Nucleofector® System HPRT1 38228-S

No EnhancerEquimolar Enhancer3 µM Enhancer

Cpf1 ribonucleoprotein complex concentration (µM)

T7E

I tot

al e

ditin

g ef

ficie

ncy

(%)

20

0 1 2 3 4 5 60

10

20

30

40

50

60

70

80

90

100

HEK 293—RNP—Amaxa® Nucleofector® SystemHPRT1 38330-AS

No EnhancerEquimolar Enhancer3 µM Enhancer

Cpf1 ribonucleoprotein complex concentration (µM)

T7E

I tot

al e

ditin

g ef

ficie

ncy

(%)

** = Toxicity

Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector®

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0 1 2 3 4 5 60

10

20

30

40

50

60

70

80

90

100

HEK 293—RNP—Neon® Transfection SystemHPRT1 38330-AS

No EnhancerEquimolar Enhancer1.8 µM Enhancer

Cpf1 ribonucleoprotein complex concentration (µM)

T7EI

tota

l edi

ting

effici

ency

(%)

Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Neon®

**

* = Toxicity

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Alt-R™ CRISPR-Cpf1 RNP editing efficiency—Nucleofector® vs. Neon® electroporation

38115-AS 38186-S 38228-S 38330-AS0102030405060708090

100

HEK 293 Cells

NFXNNeon

HPRT1 crRNA location and guide strand

T7EI

tota

l edi

ting

efficie

ncy

(%)

5 µM RNP3 µM Enhancer

5 µM RNP1.8 µM Enhancer

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Alt-R™ Cpf1 Electroporation Enhancer does not alter indel profile

Indel Size Distribution - NGS - HEK293

Indel size (bp)-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10

Sequ

ence

s (%)

0

5

60

80

1000 µM Cpf1 Electroporation Enhancer3 µM Cpf1 Electroporation Enhancer

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Alt-R™ CRISPR-Cpf1 editing—time course

15 25 35 45 55 65 750102030405060708090

100

Time post-transfection (hr)

T7E

I tot

al e

ditin

g ef

ficie

ncy

(%)

15 25 35 45 55 65 750102030405060708090

100

38115-AS38186-S38228-S38330-AS

Time post-transfection (hr)

HEK293 HeLa5 µM RNP—3 µM Enhancer—Amaxa® Nucleofector ® System

T7E

I tot

al e

ditin

g ef

ficie

ncy

(%)

25

Flanking arm

length

927257473727

Cpf1 cleavage

crRNA guide

GAATTC(EcoRI)

Total lengthssODN

190

150

120

100

80

60

Homology-directed repair in HEK 293–Cpf1 stable cell line

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Homology-directed repair in HEK 293–Cpf1 stable cell line

190

150

120

100 80 60 190

150

120

100 80 60

Non-targeted strand (nt) Targeted strand (nt)

0

20

40

60

80

100

HEK 293—HPRT1 38343-SRNAiMAX®—30 nM crRNA—30 ng HDR template (ssODN)

EcoRIT7

T7EI

tota

l edi

ting

effici

ency

(%)

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Alt-R™ CRISPR-Cpf1 RNP delivery using lipofection

1:1 2:1 5:1 1:1 2:1 5:1 1:1 2:1 5:110 nM Cpf1 30 nM Cpf1 50 nM Cpf1

0102030405060708090

100

HEK 293—RNAiMAX™

Cpf1 concentration and ratio crRNA:Cpf1

T7EI

tota

l edi

ting

effici

ency

(%)

* = Toxicity

* * * *

*

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Positive controls for genome editing using the Alt-R™ CRISPR-Cpf1 System

Hs - HEK 293 Mm - Hepa1-6 Rn - RAT20102030405060708090

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5 µM RNP—3 µM EnhancerAmaxa® Nucleofector ® System

T7EI

tota

l edi

ting

effici

ency

(%)

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Comparison of genome editing using SpCas9 vs. AsCpf1

* Molecular weight of Alt-R™ Nuclease

† N = any base V = A, C, or GTTTV

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Alt-R™ CRISPR-Cpf1 System

Core components:• Alt-R CRISPR-Cpf1 crRNA• Alt-R A.s. Cpf1 Nuclease 2 NLS• Alt-R Cpf1 Electroporation EnhancerSequences 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.

THANK YOU

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