High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’...
Transcript of High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’...
![Page 1: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/1.jpg)
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
![Page 2: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/2.jpg)
Alternative CRISPR nucleases: A.s. Cas12a (Cpf1)
2
• ~1300 AA, smaller than Cas9
• Single 41-44mer RNA trigger
• “TTTV” PAM site – expands sequence space for editing vs. “NGG”
![Page 3: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/3.jpg)
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
![Page 4: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/4.jpg)
Optimize Cas12a crRNA length and test for
compatibility with chemical modification
4
![Page 5: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/5.jpg)
AsCas12a crRNA protospacer length optimization
5
0
10
20
30
40
50
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
![Page 6: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/6.jpg)
AsCas12a crRNA 2’OMe testing
6
Residues affected by 2’OMe modification
![Page 7: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/7.jpg)
AsCas12a crRNA modification tolerance map
7
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
![Page 8: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/8.jpg)
0
10
20
30
40
50
60
70
80
90
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
8
“TTTV” not “TTTN”
233 Cas12a RNPs delivered
using electroporation into
HEK293 cells that target
multiple genes
![Page 9: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/9.jpg)
Cas12a on-target efficiency adjusted for TTTV PAM site
9
![Page 10: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/10.jpg)
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
10
![Page 11: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/11.jpg)
Improvement in Cas12a activity with protein engineering
11
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
![Page 12: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/12.jpg)
Bacterial mutagenesis primary screen
• Cas12a activity in human cells is relatively low
• TTTT PAM sequences are rarely cut by Cas12a
12
TTTC TTTT- toxin + toxin - toxin + toxin
![Page 13: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/13.jpg)
Isolation of an AsCas12a mutant with increased activity
13
![Page 14: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/14.jpg)
14
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
![Page 15: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/15.jpg)
Cell-free validation of AsCas12a mutant phenotypes
15
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
![Page 16: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/16.jpg)
Cas12a v4 Functional Performance
16* = TTTT PAM
![Page 17: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/17.jpg)
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
17
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 (
%)
![Page 18: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/18.jpg)
Summary of AsCas12a improvement studies
18
![Page 19: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/19.jpg)
19
Will the same changes also improve LbCas12a?
![Page 20: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/20.jpg)
Other ways to improve Cas12a function as a genome editing tool
20
• Electroporation Enhancer
• HDR Enhancer
![Page 21: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/21.jpg)
0
10
20
30
40
50
60
70
80
90
100
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
21
* * = Toxicity
Like Cas9, Cas12a benefits from ssDNA “electroporation enhancer”
![Page 22: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/22.jpg)
22
Ultramer ssODN designed to insert 6 bp EcoRI recognition site
HDR Enhancer present in final incubation media (media change after 24 hours)
0
10
20
30
40
50
60
70
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
![Page 23: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/23.jpg)
23
Ultramer ssODN designed to insert 6 bp EcoRI recognition site
Increased nuclease activity = increased HDR efficiency
![Page 24: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/24.jpg)
24
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
![Page 25: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/25.jpg)
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
25
![Page 26: High efficiency genome editing using a novel mutant AsCas12a · UgUAGAu/nnnnnnNNNNNNnNNnNNNNN 3’ U ||||| C ||||| uCAUCUUuaaU 5’ For routine applications, simple end-block works](https://reader035.fdocuments.in/reader035/viewer/2022070916/5fb66b5c87830d21b1154692/html5/thumbnails/26.jpg)
Thanks to the scientists who contributed to these studies …
26
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