Rolf Turk PhD, Staff Scientist Integrated DNA Technologies Alt-R™ CRISPR-Cas9 System ribonucleoprotein delivery and optimization 1
Rolf Turk PhD, Staff ScientistIntegrated DNA Technologies
Alt-R™ CRISPR-Cas9 System ribonucleoprotein delivery and optimization
1
Outline• Ribonucleoprotein complex
– Formation– Stability– Activity
• RNP delivery using lipofection
• RNP delivery using electroporation
2
CRISPR genome editing
DNA incorporation or gene knockout• Homology directed repair
vs. non-homologous end joining
3
Background information on IDT website
4
Implementing CRISPR/Cas9 gene editing
6
IDT CRISPR product offerings
3-step transfection using Alt-R™ CRISPR-Cas9 System
7
+
+
gRNA complex formation
RNP complex formation
RNP delivery
Step 1
Step 2
Step 3
15 minutes
10–20 minutes
30–60 minutes
T7EI mismatch detection to assay gene disruption
8
The Alt-R™ CRISPR-Cas9 System outperforms other methods
9
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
10
Majority of crRNAs have high editing efficiency
We recommend testing 3 crRNA sites per target gene for a high probability of successful, on-target editing on your first attempt.
11
crRNA design
Generating the Cas9 ribonucleoprotein complex
12
crRNA
tracrRNA
gRNA complex
crRNA:tracrRNA1:1
RNP:gRNA complex1:1 (lipo)
1:1.2 (electro)Cas9
gRNA complex
RNP
Ribonucleoprotein ease of use—highly stable Cas9 system• Storage of Alt-R™ S.p. Cas9 Nuclease 3NLS
– 60 µM stock (recommend storage at –20C)– Dilute down to 1 µM in:
• Opti-MEM® Media (Thermo Fisher)• Cas9 buffer (20 mM HEPES, 150 mM KCl)• PBS
– Store diluted Cas9 nuclease at 4C for up to 1 month
• Storage of ribonucleoprotein– Can be pre-complexed at 1 µM in:
• Opti-MEM® Media (Thermo Fisher)• Cas9 Buffer (20 mM HEPES, 150 mM KCl)• PBS
13
Alt-R S.p. Cas9 Nuclease 3NLS164 kDa60 µM = 60 pmol/µL = 10 µg/µL
14
HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8Control -80°C -20°C 4°C
0
10
20
30
40
50
60
70
80
90
100
Cas9 RNP Transfection HEK293, 10 nM RNP, 1.2 µL RNAiMAX®
Cas9 Buffer OptiMEM PBS
HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8 HPRT Site 6 HPRT Site 8
T7 E
ndon
ucle
ase
I cle
avag
e (%
)Cas9 ribonucleoprotein complex stability — 2 weeks
Control
–80˚C –20˚C 4˚C
Delivery of Cas9 ribonucleoprotein
15
Lipofection• Mix ribonucleoprotein with
cationic lipids• Incubate (time sensitive)• Apply to cells
http://www.nature.com/scitable/topicpage/what-is-a-cell-14023083
Electroporation• Hard to transfect cells, primary
cells• Mix RNP with cells in
electroporation solution• Electroporate
16
HPRT Site 4 HPRT Site 6 HPRT Site 80
10
20
30
40
50
60
70
80
90
100
IDT Supplier A Supplier B Supplier C Supplier E Supplier D
T7EI
edi
ting
efficie
ncy
(%)
HPRT site 4
HPRT site 8
HPRT site 6
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
Protein comparison—S. pyogenes Cas9 nucleaseCas9 RNP transfection, HEK293, 10 nM RNP, 1.2 µL RNAiMAX®
Gene editing—know your target(s)
17
• Targeting locus• Multiple transcript isoforms• SNPs present in your protospacer• Ploidy of cells• Known phenotype?• Will knockout lead to lethality?• Before monoclonal selection, check batch
Cas9 protein is rapidly degraded—less off-target effects
18Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 208:44–53.
Off-target indel productionWestern blot
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
HPRT Site 6 - Cas9 Cells HPRT Site 6 - RNPHPRT Site 8 - Cas9 Cells HPRT Site 8 - RNP
Time post-transfection (hrs.)
T7EI
Cle
avag
e (%
)
HEK293 Cas9 cells: 30 nM, 0.75 µL RNAiMAX®
HEK293 RNP: 10 nM, 1.2 µL RNAiMAX®
On-target editing — time course study
19
10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)
T7 E
ndon
ucle
ase
I cle
avag
e (%
)HPRT Site 6
HPRT Site 8
HPRT Site 6 – Cas9 cells
HPRT Site 8 – Cas9 cells
HPRT Site 6 – RNP
HPRT Site 8 – RNP
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
HPRT Site 6 - Cas9 Cells HPRT Site 6 - RNPHPRT Site 8 - Cas9 Cells HPRT Site 8 - RNP
Time post-transfection (hrs.)
T7EI
Cle
avag
e (%
)
HEK293 Cas9 cells: 30 nM, 0.75 µL RNAiMAX®
HEK293 RNP: 10 nM, 1.2 µL RNAiMAX®
On-target editing — time course study
20
10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)
48 hrsT7 E
ndon
ucle
ase
I cle
avag
e (%
)HPRT Site 6
HPRT Site 8
HPRT Site 6 – Cas9 cells
HPRT Site 8 – Cas9 cells
HPRT Site 6 – RNP
HPRT Site 8 – RNP
21
0
10
20
30
40
50
60
70
80
90
100
HEK293-Cas9 Cells HEK293 cells + Ribonucleoprotein Complex
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
HPRTSite 1
HPRTSite 2
HPRTSite 3
HPRTSite 4
HPRTSite 5
HPRTSite 6
HPRTSite 7
HPRTSite 8
HPRTSite 9
HPRTSite 10
HPRTSite 11
HPRTSite 12
10 nM RNP150 µL volume= 1.5 pmol (0.25 µg)
HEK293 Cas9 Cells: 30 nM, 0.75 µL RNAiMAX®
HEK293 RNP: 10 nM, 1.2 µL RNAiMAX ®
RNP transfection leads to maximal editing
Comparing sources of S. pyogenes Cas9 nuclease
22
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
23
0
10
20
30
40
50
60
70
80
90
100
T7EI
Cle
avag
e (%
)T7
End
onuc
leas
e I
cleav
age
(%)
Unmod Alt-R™ crRNA—Cas9 cells
Unmod Alt-R™ crRNA—RNP Modified Alt-R™ crRNA—RNP
CCR5Site 1
CCR5Site 2
CCR5Site 3
CCR5Site 4
CCR5Site 5
CCR5Site 6
CCR5Site 7
CCR5Site 8
CCR5Site 9
CCR5Site 10
CCR5Site 11
CCR5Site 12
HEK293 Cas9 Cells: 30 nM, 0.75 µL RNAiMAX®
HEK293 RNP: 10 nM, 1.2 µL RNAiMAX ®
Modified Alt-R™ crRNA:tracrRNA + Cas9 protein (RNP)
Complete experimental instructions can be found in the User Guide PDF at www.idtdna.com/CRISPR, under the Support tab.
Experimental outline for the Alt-R™ CRISPR-Cas9 System
24
Transfection optimization
25RN
P LIPIDSEDITING
TYPE
VIABILITY
CELLS
Transfection optimization
26RN
P
LIPIDS
EDITINGTY
PE
VIABILITY
CELLS
27
Toxicity present
Lipofec-tamine® RNAiMAX
Lipid A Lipid B Lipid C Lipid D Lipid E Lipid F Lipid G0
10
20
30
40
50
60
0.25 µL 0.5 µL 0.75 µL 1.0 µL 1.25 µL 1.5 µL
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
Cas9 RNP transfection, HEK293, 10 nM RNP
Lipid optimization—Cas9 RNP transfection
Optimization strategy for lipofection• Follow recommendations from Alt-R™ CRISPR-Cas9 System user
manual• Include positive and negative controls• Pay close attention to cell viability• Use low passage numbers• Cell density should be sub-confluent
• If optimization is necessary:– Test a range of RNP concentrations– Test different amounts of cells per lipofection– Test RNP to cationic lipid ratios
• Test alternatives to lipofection electroporation28
Delivery of RNP by electroporationAmaxa Nucleofector™ (Lonza)• 1-, 16-, or 96-well format• Online database
– Cell lines– Primary cells
• Expensive• ‘Black box’
29
Neon™ Transfection System (Thermo Fisher)• Known parameters• Easy optimization• Online database
– Cell lines
• Expensive• Single cuvette• No recommendations
What to look for when doing electroporation • Requires higher ribonucleoprotein concentrations (2–4 µM)• Cas9 ribonucleoprotein complex is not encapsulated
– More accessible to nucleases– More accessible to proteinases
• Electroporation parameters differ per cell line– Amaxa Nucleofector
• Cell database• Optimization protocol (96-well format)
– Neon Transfection System• Optimization protocols
30
Optimization strategy for electroporation• Find optimal electroporation parameters• Follow recommendations from IDT User Manual• Include positive and negative controls• Addition of Carrier DNA to boost editing efficiency• Use low passage numbers• Cell density should be sub-confluent
• Pay close attention to cell viability• If necessary, test a range of RNP concentrations• Test different amounts of cells per electroporation
31
32
0.0 2.0 4.0 6.0 8.0 10.00
10
20
30
40
50
60
70
80
90
100
HPRT Site 6 HPRT Site 8
Ribonucleoprotein complex (µM)
T7EI
Edi
ting
Effici
ency
(%)
3.3 µM RNP30 µL volume= 100 pmol (16 µg)
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
HPRT Site 6
HPRT Site 8
Nucleofection — Effect of RNP concentration on editing efficiency
Cas9 RNP, HEK293
33
0 µM
1 µM
4 µM
10 µ
M
1 µM
4 µM
10 µ
M
1 µM
4 µM
10 µ
M
1 µM
4 µM
10 µ
M
No Carrier
20 nt 95 nt 200 nt tRNA
0
5
10
15
20
25
30
35
T7EI
Edi
ting
Effici
ency
(%) 3.3 µM RNP
30 µL volume= 100 pmol (16 µg)
T7 E
ndon
ucle
ase
I cle
avag
e (%
)RNP Nucleofection — Effect of carrier DNA properties on editing efficiency
Cas9 RNP, HEK293
34
Opt
_01
Opt
_02
Opt
_03
Opt
_04
Opt
_05
Opt
_06
Opt
_07
Opt
_08
Opt
_09
Opt
_10
Opt
_11
Opt
_12
Opt
_13
Opt
_14
Opt
_15
Opt
_16
Opt
_17
Opt
_18
Opt
_19
Opt
_20
Opt
_21
Opt
_22
Opt
_23
Opt
_24
0.1
µM 0
.3 µ
M1
µM3.
3 µM
5 µM
10 µ
M
Neon Electroporation Nucleofection
0
10
20
30
40
50
60
70
80
T7EI
Cle
avag
e (%
)
low high
Cell viability
Optimization of Neon System—Alt-R™ CRISPR-Cas9 System RNP
1.5 µM RNP10 µL volume= 18 pmol (3 µg)
T7 E
ndon
ucle
ase
I cle
avag
e (%
)
Cas9 RNP, HEK293
Advantages of Alt-R™ CRISPR-Cas9 System
35
• Guide RNA complex using Alt-R™ CRISPR crRNA and tracrRNA• Quality controlled manufacturing• Ease of use• Modifications for prolonged stability in cellular
environments• Reduced toxicity from innate immune stimulation
• Alt-R™ S.p. Cas9 Nuclease 3NLS• Ease of use• Controlled delivery• Fewer off-target effects• No toxicity• Cost effective
The Alt-R CRISPR Cas9 System
36
A complete system• Alt-R™ S.p. Cas9 Nuclease 3NLS• Alt-R™ CRISPR crRNA• Alt-R™ CRISPR tracrRNA• Alt-R™ CRISPR Control Kits (Human, Mouse, Rat)
Delivered as ribonucleoprotein• By cationic lipids:
- Lipofectamine® RNAiMAX® (Thermo Fisher)- Lipofectamine® CRISPRMAX™ (Thermo Fisher)
• By electroporation- Nucleofector™ (Lonza)- Neon™ system (Thermo Fisher)
Visit www.idtdna.com/CRISPR for more information• Support tab
– User guide and short protocols– Previous Alt-R™ CRISPR-Cas9
System webinars– Short tutorial videos on how to
order• Performance tab
– View key data from Alt-R™ CRISPR-Cas9 System experiments
Alt-R™ CRISPR-Cas9 System additional resources
37
“Best tech support ever,
@idtdna!”
Questions?TALK TO A PERSON.
Lauren SakowskiOur 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.comOr email: [email protected]
THANK YOU!We will email you the webinar recording and slides next week.