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High efficiency genome editing using a
novel mutant AsCas12a
Mark Behlke MD, PhD
Chief Scientific Officer
1
February 27, 2019 CRISPR in Drug Discovery 2019 Oxford
Alternative CRISPR nucleases: A.s. Cas12a (Cpf1)
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• ~1300 AA, smaller than Cas9
• Single 41-44mer RNA trigger
• “TTTV” PAM site – expands sequence space for editing vs. “NGG”
Like Cas9, Cas12a OTEs are reduced using RNP compared to plasmid
3Kim et al., Nature Biotech, 2016
OTE plasmidOTE RNP
background
Cas12a intrinsically has
lower OTEs than Cas9
Optimize Cas12a crRNA length and test for
compatibility with chemical modification
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AsCas12a crRNA protospacer length optimization
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0
10
20
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40
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60
70
80
90
100
38171-AS 38254-AS 38325-S 38337-AS 38351-S 38538-AS
T7
EI E
dit
ing (
%)
HPRT1 crRNA location and guide strand
HEK293-Stable-Cas12a – 30 nM crRNA
22 mer
21 mer
19 mer
18 mer
17 mer
21 = 20 > 19 >> 18
AsCas12a crRNA 2’OMe testing
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Residues affected by 2’OMe modification
AsCas12a crRNA modification tolerance map
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UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’
U |||||
C |||||
uCAUCUUuaaU 5’
For routine applications,
simple end-block works as well as
expensive, complex modification patterns
Single 2’OMe base
sensitivity
2’OMe-modified bases = ACUG
2’F-modified bases = ACUG
RNA = acug
Blocks of
modifications
0
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100
0 20 40 60 80
T7
EI To
tal E
dit
ing E
ffic
ien
cy
(%)
TTTA
TTTC
TTTG
TTTT
Cas12a editing efficiency is highly PAM-site dependent
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“TTTV” not “TTTN”
233 Cas12a RNPs delivered
using electroporation into
HEK293 cells that target
multiple genes
Cas12a on-target efficiency adjusted for TTTV PAM site
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Rationale and strategy for improving AsCas12a
• Problems with AsCas12a
– AsCas12a enzymatic activity < SpCas9
– TTTV is too restrictive
• Strategies to improve AsCas12a properties
– Protein engineering – optimize linkers, NLS, other features
– Primary bacterial mutagenesis screen
• Random low-fidelity PCR mutagenesis of a broadly targeted region
– Secondary focused saturation mutagenesis screen
• Comprehensive mutagenesis of critical AA positions from primary screen
– Results coupled to NGS
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Improvement in Cas12a activity with protein engineering
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0
10
20
30
40
50
60
70
80
90
100
T7
EI C
lea
va
ge
(%
)
Rank Order
Cas12a V1 - Replicate 1
Cas12a V1 - Replicate 2
Cas12a V3 - Replicate 1
Cas12a V3 - Replicate 2
Improved activity, still TTTV PAM
Bacterial mutagenesis primary screen
• Cas12a activity in human cells is relatively low
• TTTT PAM sequences are rarely cut by Cas12a
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TTTC TTTT- toxin + toxin - toxin + toxin
Isolation of an AsCas12a mutant with increased activity
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Deep-characterization of libraries by NGS
5’ 3’
Mutagenized region
Mutant 1
Mutant 3
Mutant 2
Position
AsCas12a
2 AA positions
were “hot spots”
in the screen
Cell-free validation of AsCas12a mutant phenotypes
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0 4 0 8 0 1 2 0
0
2 0
4 0
6 0
8 0
1 0 0
3 0 0 2 3 0 0 4 3 0 0
W T - T T T T
T im e (s )
% o
f D
NA
cle
av
ag
e
0 4 0 8 0 1 2 0
0
2 0
4 0
6 0
8 0
1 0 0
3 0 0 2 3 0 0 4 3 0 0
M u ta n t 3 - T T T T
T im e (s )
% o
f D
NA
cle
av
ag
e
0 4 0 8 0 1 2 0
0
2 0
4 0
6 0
8 0
1 0 0
3 0 0 2 3 0 0 4 3 0 0
M u ta n t 1 - T T T T
T im e (s )
% o
f D
NA
cle
av
ag
e
0 4 0 8 0 1 2 0
0
2 0
4 0
6 0
8 0
1 0 0
3 0 0 2 3 0 0 4 3 0 0
M u ta n t 1 + 3 - T T T T
T im e (s )
% o
f D
NA
cle
av
ag
e
Mutant 2 - TTTTWT - TTTT Mutant 2 - TTTT
Mutant 1 - TTTT Mutant 1+2 - TTTT
New v4 Cas12a
Cas12a v4 Functional Performance
16* = TTTT PAM
Cas12a v
1
Cas12a v
3
Mu
tan
t 1
Mu
tan
t 2
Cas12a v
4
2 0
4 0
6 0
8 0
1 0 0
T7
EI
Cle
av
ag
e (
%)
Cas12a v
1
Cas12a v
3
Mu
tan
t 1
Mu
tan
t 2
Cas12a v
4
2 0
4 0
6 0
8 0
1 0 0
T7
EI
Cle
av
ag
e (
%)
Cas12a v4 cleaves TTTN with high efficiency in human cells
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TTTN TTTT TTTV
Cas12a v
1
Cas12a v
3
Mu
tan
t 1
Mu
tan
t 2
Cas12a v
4
2 0
4 0
6 0
8 0
1 0 0
T7
EI
Cle
av
ag
e (
%)
Summary of AsCas12a improvement studies
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Will the same changes also improve LbCas12a?
Other ways to improve Cas12a function as a genome editing tool
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• Electroporation Enhancer
• HDR Enhancer
0
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0 1 2 3 4 5 6
T7
EI to
tal e
ditin
g e
ffic
ien
cy (
%)
Cas12a ribonucleoprotein complex concentration (µM)
HEK 293—RNP—Amaxa® Nucleofector® SystemHPRT1 38330-AS
No Enhancer
Equimolar Enhancer
3 µM Enhancer
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* * = Toxicity
Like Cas9, Cas12a benefits from ssDNA “electroporation enhancer”
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Ultramer ssODN designed to insert 6 bp EcoRI recognition site
HDR Enhancer present in final incubation media (media change after 24 hours)
0
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MET TNPO3 site1 TNPO3 site 2 HPRT control HPRT site 1 HPRT site 2 HPRT site 3 HPRT site 4
Alt-R S.p. Cas9 nuclease v3 Alt-R A.s. Cas12a (Cpf1) nuclease v3
Eco
RI c
leav
age
(%
)HDR Enhancer improves HDR efficiency for both Cas9 and Cas12a (Cpf1) nucleases
Neon electroporation, Jurkat cells - 3 µM ssODN
No treatment DMSO 30 µM HDR Enhancer
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Ultramer ssODN designed to insert 6 bp EcoRI recognition site
Increased nuclease activity = increased HDR efficiency
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Availability
Alt-R® A.s. Cas12a (Cpf1) v3 Catalog item
Alt-R® A.s. Cas12a (Cpf1) Ultra v4 May
Beta test samples can be obtained from IDT R&D now
Conclusions• WT AsCas12a editing in mammalian cells is inherently less robust than SpCas9
– More restrictive PAM (TTTV as opposed to NGG)
– Slightly lower enzymatic activity (more of an issue for RNP than plasmid/viral)
• Protein engineering can significantly improve performance at TTTV sites
• Directed evolution efforts led to a mutant variant (Alt-R AsCas12a V4) with
superior properties
– Higher overall editing efficiencies
– Enabled TTTT PAM cleavage
• Contrary to conventional wisdom, high HDR rates can be achieved using Cas12a;
new Cas12a mutant and use of “HDR Enhancer” helps
• Like Cas9, “Electroporation Enhancer” improves RNP activity
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Thanks to the scientists who contributed to these studies …
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Integrated DNA Technologies
– Ashley Jacobi
– Garrett Rettig
– Mollie Schubert
– Rolf Turk
– Bernice Thommandru
– Matt McNeill
– Michael Christodoulou
– Chris Vakulskas
– Michael Collingwood
– Nicole Bode
– Sarah Beaudoin
– Liyang Zhang
Coralville, Iowa
USA
CRISPR Protein Team
