Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery. In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.

1. Increasing genome editing efficiency with
optimized CRISPR-Cas enzymes
Christopher Vakulskas, PhD
Senior Staff Scientist
1

2. Outline: Using improved (V3) CRISPR enzymes
• The basics of genome editing
• Improving genome editing with RNP delivery
– RNP mitigates toxicity and off-target effects
– Alt-R HiFi Cas9 dramatically reduces off-target effects
• Improved (V3) Cas9 Nucleases
• Alternative CRISPR nucleases: Cas12a (Cpf1)
– Overview
– V3 improvements
2

3. Genome editing
3

4. CRISPR-Cas9 genome editing
• RNA-guided endonuclease
• 20 nt protospacer
• PAM site (NGG)
• Native two-part crRNA and tracrRNA or one piece sgRNA
4
Alt-R crRNA:tracrRNA ribonucleoprotein (RNP) complex

5. Cas9 sgRNA vs. crRNA:tracrRNA complexes
crRNA:tracrRNA complex
• Not ideal to express from DNA expression
vectors
• Not ideal for IVT
• Efficient for chemical synthesis
– 20 bases unique, 16 bases constant
– 67 bases universal tracrRNA
• Easy to modify, escape immune response
5
sgRNA (single guide RNA)
• Ideal for DNA expression cassettes
• Ideal for IVT (low cost)
• Inefficient for chemical synthesis
– 20 bases unique, 80 bases constant
– Hence, higher cost
• Costly to modify, induces immune
response, and IVTs cannot be modified

6. Transfection of IVT sgRNAs can be toxic to cells
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• Successful gene editing
• Transfection of IVT sgRNAs can result in:
– Large scale cell death
– Induction of innate immune response
HEK-293 cells only 30 nM sgRNA IVT 30 nM 2-part RNA

7. IVT sgRNAs trigger immune response, synthetic 2-part RNA oligos do not
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• IFITM1, RIGI, and OAS2 had similarly high induction when treated with IVT sgRNA
(triphosphate removed)
• No inductions were detected when treated with synthetic 2-part gRNA complexes
Hs SFRS9 qPCR assay (normalizer) Hs IFIT1 qPCR assay

8. Implementing CRISPR-Cas9 gene editing
8

9. 3-step transfection using Alt-R CRISPR-Cas9 RNP System
9
+
+
gRNA complex formation
RNP complex formation
RNP delivery
Step 1
Step 2
Step 3
15 minutes
10 minutes
30–60 minutes
1:1
1:1
Lipofection: 10 nM
Electroporation: 1–3 µM
Microinjection
Cas9

10. 10
On-target site
Empirically determined
off-target sites
Tsai SQ, Zheng Z, et al. (2015) GUIDE-seq enables
genome-wide profiling of off-target cleavage by CRISPR-
Cas nucleases. Nat Biotechnol, 33(2):187–197.
S.p. Cas9 is likely to produce off-target cleavage
(particularly with plasmid expressed sgRNA and Cas9)

11. Ratio of on/off target editing depends heavily on Cas9 source
(i.e., plasmid, mRNA, or protein)
11
Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian
cell engineering via Cas9 protein transfection. J Biotechnol, 208:44–53.

12. RNP delivery of wild-type Cas9 reduces off-target editing
12
GAGTCCGAGCAGAAGAAGAAGGG EMX1 on-target site
GAGTTAGAGCAGAAGAAGAAAGG Off-target site 1
GAGTCTAAGCAGAAGAAGAAGAG Off-target site 2
0
10
20
30
40
50
60
70
80
90
100
Low-level constant
expression
4 µM 2 µM 1 µM 0.5 µM
HEK293-Cas9
Cells
WT Alt-R S.p. Cas9 RNP
IndelbyNGS(%)
On target
Off target 1
Off target 2

13. Guide RNA algorithms provide predictions for Cas9
off-target effects
• Extremely challenging to accurately predict Cas9 off-target sites
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CRISPR Design SVM (MIT) CCTop Target SVM (Heidelberg University)

14. NGS analysis of “cell-free” Cas9 cleavage sites
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0
50
100
150
200
250
300
350
Cleavagefrequency(NGSreads)
On target Off target SVM-Predicted off-target sites
Predicted Not predicted
9%
AR-S-1893
GTTGGAGCATCTGAGTCCAGGGG

15. Cas9 off-target effects
• Delivery of Cas9 RNP complex reduces off-target editing, but it is not
a total solution
• Other solutions to reduce OTE have significant drawbacks
– crRNA length reduction (18–19 nt)
– Chemical modification
• What about high-fidelity Cas9 proteins?
15

16. Published, rationally-designed, high-fidelity Cas9 mutants
• eSpCas9 (1.1)
– Slaymaker IM, Gao L, et al. (2016)
Rationally engineered Cas9 nucleases
with improved specificity. Science,
351(6268):84–88.
• SpCas9-HF1
– Kleinstiver BP, Pattanayak V, et al. (2016)
High-fidelity CRISPR–Cas9 nucleases
with no detectable genome-wide off-target
effects. Nature, 529(7587):490–495.
16

17. Existing high-fidelity Cas9 mutants are ill-suited for
the RNP delivery method
17
0
10
20
30
40
50
60
ON OFF ON OFF ON OFF
EMX1 HEKSite4 VEGFA3
T7EICleavage(%)
WT Cas9 eSpCas9(1.1) SpCas9-HF1
****
ns
**** ****
****
****
****
****
****
Literature-derived gRNAs Internal IDT gRNAs

18. RNP delivery of existing high-fidelity Cas9
• Protein mutations were selected based on plasmid delivery results
– Continued and long-lasting Cas9 synthesis
– Plasmid delivery prone to toxicity and immune stimulation
• No existing Cas9 HiFi mutant that works well as RNP
– Reduced off-target editing at the expense of on-target potency
• Proprietary bacterial selection system for HiFi Cas9 mutants
– Double selection for mutants that avoid off-target editing but have
successful cleavage of the intended on-target site
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19. Bacterial screen to identify novel high-fidelity Cas9 mutants
19

20. Alt-R S.p. HiFi Cas9 Nuclease 3NLS: On-target
performance is significantly improved
20
Alt-R CRISPR-Cas9 System
10 nM RNP, Lipofection
HPRT locus
HEK-293 cells
Chemically modified sgRNAs
Amaxa® Nucleofector® (Lonza)
CD34+ HSPCs
0
20
40
60
80
100
HBB CCR5 HEXB TRAC
IndelbyNGS(%)
WT Cas9
eSpCas9
SpCas9-HF1
Alt-R HiFi Cas9
0
10
20
30
40
50
60
70
80
90
T7EIcleavage(%)
WT Cas9
eSpCas9
SpCas9-HFI
Alt-R HiFi Cas9
Danny Dever
Matt Porteus
Stanford University

21. Comparison of properties of WT Cas9 and improved fidelity Cas9 mutants
21
Single OTE site studied using PCR/EMCA method
T7EIcleavage(%)

22. Analyzing off-target editing globally
• Analyzing known off-target sites with PCR and T7EI is an imprecise
method for investigating reduced off-target editing
• We utilized a two-step approach to detect global off-target editing
– All edited sites were detected using the GuideSEQ procedure
– Amplicon-based NGS with rhAmp primer chemistry was used to calculate
indel frequency at every site detected with GuideSEQ
• We analyzed global editing with this technique using 4 different
crRNAs that target the AR, EMX1, HBB, and HPRT loci in HEK293
cells
22

23. GUIDE-Seq screen: WT vs. IDT HiFi Cas9 in HEK-293 cells
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24. Alt-R HiFi Cas9 reduces global off-target editing
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On-target editing Off-target editing
EMX1 - GAGTCCGAGCAGAAGAAGAAGGG

25. 25

26. 1
10
100
-20 -19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
IndelbyNGS(%) Alt-R S.p. Cas9 Alt-R S.p. HiFi Cas9 Cells Only
Alt-R HiFi Cas9 does not alter the expected indel profile
26
0
insertionsdeletions
• HPRT-targeting crRNA delivered into HEK-293 cells by electroporation
• Insertion and deletion profile analyzed at the expected on-target site by NGS

27. HDR experiments with Alt-R S.p. HiFi Cas9
• HiFi Cas9 mediates HBB modification in
HSPCs transduced with an ssAAV6 donor
• Off-target editing is significantly reduced at
all doses
• Modest reduction in on-target HDR rate
can be compensated with increased
enzyme without sacrificing reduction in off-
target editing
27

28. Ex-vivo genome editing will be an early medical application
for Alt-R S.p. HiFi Cas9
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29. Performance of upgraded Cas9 (V3) nuclease
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0
10
20
30
40
50
60
70
80
90
100
IndelbyNGS(%)
Alt-R S.p. Cas9
Alt-R S.p. Cas9 V3
12 crRNAs targeting the HPRT locus

30. Alt-R (V3) Cas9 Nuclease upgrades improve performance
30
4 µM RNP Nucleofection – 1:1:1 (Cas9:crRNA:tracrRNA) – 2 µM Cas9 Electroporation Enhancer - HEK293 cells
EMX1 - GAGTCCGAGCAGAAGAAGAAGGG

31. Alt-R CRISPR-Cas9 Summary
• RNP delivery format mitigates off-target effect risk and immune
system stimulation common with plasmid-based delivery methods
• Alt-R Cas9 protein delivered as RNP reduces many but not all
problematic off-target sites
• Alt-R HiFi Cas9 protein significantly reduces the vast majority of off-
target cleavage events
• Improved suite of (V3) Cas9 nucleases increase editing efficiency in
live cells
31

32. Improved (V3) Cas9 nucleases
• Alt-R S.p. Cas9 V3 nuclease
– 100, 500, and 5000 µg quantities
• Alt-R S.p. HiFi Cas9 V3 nuclease
– 100, 500, and 5000 µg quantities
• Alt-R S.p. dCas9 V3 protein
– Quality controlled to:
• Ensure no nuclease activity
• Confirm retention of specific RNA-guided DNA binding activity
• Alt-R S.p. Cas9 D10A and H840A V3 nickases
– Targeted cleavage of a single DNA strand
– Previous IDT Webinar entitled “Optimized methods to use Cas9 nickases in genome editing”
(www.idtdna.com/webinars)
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33. Improved Alt-R Cas12a (Cpf1) V3 nuclease
33

34. Alternative CRISPR nuclease – A.s. Cas12a
• RNA-guided endonuclease
• Cas12a: CRISPR from Prevotella and Francisella 1
• Class II, type V
• Cas12a 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
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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.

35. Optimizing protospacer length of Cas12a crRNA
35
0
20
40
60
80
100
38171-AS 38254-AS 38325-S 38337-AS 38351-S 38538-AS
T7EIEditing(%)
HPRT1 crRNA location and guide strand
HEK-293-stable-Cas12a – 30 nM crRNA
22 mer 21 mer 19 mer 18 mer 17 mer
A.s. Cas12a

36. Cas12a (Cpf1) OTEs are also lower using RNP compared
to plasmid
36
Kim et al., (2016) Genome-wide analysis reveals specificities of Cpf1
endonucleases in human cells. Nature Biotech, 34:863-868
OTE plasmid
OTE RNP
background
Reports suggest that
Cas12a intrinsically has
lower OTEs than Cas9.
Does this truly reflect
intrinsic lower OTEs, or
does it reflect that Cas12a
only works at 2/3 of PAM
sites, whereas Cas9 works
at most PAM sites?

37. Alt-R CRISPR-Cas12a RNP complex formation
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38. Effect of Alt-R Cas12a Electroporation Enhancer with RNP
38
0
10
20
30
40
50
60
70
80
90
100
T7EI total editing efficiency (%)
HPRT1 crRNA location and guide strand
HEK 293—5 µM RNP—Amaxa® Nucleofector® System
0 µM Enhancer
3 µM Enhancer
5 µM Enhancer

39. 0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80
T7EITotalEditingEfficiency(%)
TTTA
TTTC
TTTG
TTTT
Cas12a editing efficiency is highly PAM-site dependent
39
“TTTV” not “TTTN”
233 Cas12a RNPs delivered using
electroporation into HEK-293 cells
from multiple genes

40. Cas12a on-target efficiency adjusted for TTTV PAM site
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0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
T7EI total editing efficiency (%)
Ranked editing efficiency
STAT3: exons 5 and 6
HEK-293—RNP—Amaxa Nucleofector System
Cas12a (TTTN) Cas12a (TTTV) SpCas9 (NGG)
Continuing to test Cas12a at >1000 sites for algorithm development
and improved Cas12a site selection

41. Improved Alt-R Cas12a V3 protein increases overall editing
efficiencies
41
0
10
20
30
40
50
60
70
80
90
100
T7EICleavage(%)
Rank Order
Cas12a V1 - Replicate 1
Cas12a V1 - Replicate 2
Cas12a V3 - Replicate 1
Cas12a V3 - Replicate 2

42. Take home messages…
• RNP delivery of CRISPR-Cas9 and CRISPR-Cas12a systems
– Simplifies delivery
– Reduce complications from immune system stimulation
– Reduces off-target editing
• RNP delivery of Alt-R S.p. HiFi Cas9
– Near-WT on-target editing efficiencies at 90% of sites
– Exceptional reduction in off-target editing
• Improved (V3) CRISPR proteins
– Industry-leading
– Dramatically increase editing efficiencies in live cells
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43. THANK YOU
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