The document discusses the use of Alt-R Cas9 nickases (D10A and H840A) for genome editing, detailing techniques for designing gRNA pairs and donor templates for homology-directed repair (HDR). It emphasizes that D10A is more effective than H840A in mediating HDR and provides specific design guidelines for using these nickases to enhance editing efficiency. The authors recommend optimal distances between targeted nick sites and highlight additional considerations for successful HDR applications in different insertion contexts.

1. Optimized methods to use Cas9 nickases
in genome editing
Mollie Schubert, MS, Research Scientist
Shuqi Yan, MS, Research Scientist
1

2. Outline: Using Alt-R® Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
2

3. Outline: Using Alt-R Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
3

4. Genome editing—an expanding toolbox
4

5. CRISPR-Cas9 genome editing
• RNA-guided endonuclease
• 20 nt protospacer
• PAM site (NGG)
• Native two-part crRNA and tracrRNA, or one-piece sgRNA
5

6. CRISPR-Cas9 genome editing
• 67 nt universal tracrRNA
• 36 nt site specific crRNA
• Chemically modified to prevent
immune stimulation, nuclease
degradation
6
Alt-R crRNA:tracrRNA Alt-R sgRNA
• 100 nt site specific sgRNA
• Chemically modified to prevent
immune stimulation, nuclease
degradation

7. Alt-R CRISPR-Cas9 RNP System
7
+
+
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

8. 8
IDT and collaborator protocols available online
www.idtdna.com/crispr-cas9

9. Outline: Using Alt-R Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
9

10. Wild type Cas9 creates a double-strand break (DSB)
10
Cleaves both strands of
target DNAWild type Cas9

11. D10A Cas9 nickase cleaves the target strand
11
Cleaves the target strand
Cas9 D10A nickase has an
inactivated RuvC domain

12. H840A Cas9 nickase cleaves the non-target strand
12
Cleaves the non-target strand
Cas9 H840A nickase has an
inactivated HNH domain

13. Combined use of a Cas9 nickase with a pair of gRNAs
creates DSBs with overhangs
13
5’
3’
5’
3’
5’ overhang 3’ overhang
PAM-out PAM-in
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14. Outline: Using Alt-R Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
14

15. Double nicking with a Cas9 nickase and paired gRNA
generates a DSB
15

16. Both D10A and H840A prefer PAM-out gRNA pairs
16
0
10
20
30
40
50
60
70
80
90
100 40nt
46nt
51nt
63nt
68nt
85nt
118nt
130nt
18nt
26nt
36nt
59nt
66nt
73nt
97nt
PAM-out PAM-in
TotaleditingbyT7EI*assay(%)
gRNA pairs targeting HPRT1
HEK-293, Lipofection
D10A H840A
* T7EI — T7 endonuclease I that can be used to detect indel formation (total editing) frequency

17. Efficiency drops when the gRNAs are too close
17
0
10
20
30
40
50
60
70
80
90
100
7nt
12nt
18nt
23nt
37nt
40nt
60nt
62nt
67nt
TotaleditingbyNGS(%)
PAM-out
Cas9 D10A with gRNA pairs targeting AAVS1 locus
HEK-293, Nucleofection

18. Indel profiles vary between D10A and H840A
18
0
1
2
3
4
5
6
7
8
9
10
-100 -50 0 50 100 150
Frequencies(%)
Indel size
NGS data of H840A
(Total editing: 77.1%)
Deletion
1.3 %
Insertion
75.8 %
0
1
2
3
4
5
6
7
8
9
10
-100 -50 0 50 100 150
Frequencies(%)
Indel size
NGS data of D10A
(Total editing: 96.1%)
Deletion
69.5%
Insertion
26.6%
Target site: HPRT1 51 nt PAM-out
No HDR donor, editing evaluated using NGS

19. gRNA design recommendations for nickases
• Use gRNA pair in PAM-out orientation for both D10A and H840A
• Make sure gRNA pair is at an optimal distance
Ø D10A: targeted nick sites separated by 37–68 bp
Ø H840A: targeted nick sites separated by 51–68 bp
19

20. Outline: Using Alt-R Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
20

21. Using nickases for homology directed repair
• D10A vs H840A: which to choose?
• HDR donor design
Ø Small insertion (ssODN; IDT Ultramer® Oligonucleotides)
– What is the optimal homology arm length?
– Where to place the insert?
Ø Large insertion (long ssDNA; IDT Megamer® ssDNA Fragments)
– An example of inserting a ~800 bp fluorescent protein construct
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22. D10A is more potent than H840A at mediating HDR
22
ssODN HDR donors
Top and bottom strands
1
10
100
Top Btm Top Btm Top Btm Top Btm
D10A H840A D10A H840A
Left Right
Cleavage(%)
Compare D10A and H840A mediated
HDR at left/right nick site
EcoRI T7EI
HPRT1 51 nt PAM-out site

23. 40 nt is the optimal length of homology arms
23
HPRT1 51 nt PAM-out site
0
5
10
15
20
25
20 nt
arm
30 nt
arm
40 nt
arm
50 nt
arm
20 nt
arm
30 nt
arm
40 nt
arm
50 nt
arm
HA-E-HA HA-E-51-H-HA
HDRbyNGS(%)
ssODN arm-length walk
ssODN HDR donors
Bottom strand

24. Insertion walk away from Cas9 site(s)
24
HPRT1 51 nt PAM-out site
ssODN HDR donors
Top and bottom strands

25. Cas9 D10A is able to mediate HDR distal from the PAM site
25
0
5
10
15
20
25
30
Top Btm Top Btm Top Btm Top Btm Top Btm
Left
nick/cut
site
13 bp from
Left
25 bp from
Left
38 bp from
Left
Right nick
site (51 bp
from left)
HDR-EcoRIcleavage(%)
D10A + gRNA pair
0
5
10
15
20
25
30
Top Btm Top Btm Top Btm Top Btm Top Btm
Left
nick/cut
site
13 bp from
Left
25 bp from
Left
38 bp from
Left
Right nick
site (51 bp
from left)
HDR-EcoRIcleavage(%)
WT+left gRNA

26. 26
D10A mediates efficient HDR with larger insert
ssODN HDR donors
Top and bottom strands
HPRT1 51 nt PAM-out site
0
2
4
6
8
10
12
Btm Top Btm Top Btm Top Btm Top
Left 13 nt from
Left
25 nt from
Left
38 nt from
Left
Right
HDRbyNGS(%)
D10A + gRNA pair

27. Using Megamer ssDNA Fragments for HDR
27
T2A
////
47nt PAM-out
eGFP cell line
mCherry (711bp)
5 d
post
tfxn
• HEK-293–eGFP stable cell line
• Workflow
Day 1:
– Amaxa® (Lonza) Nucleofection:
3.5 x 106 cells,
4 µM total RNP (Cas9 D10A),
4 µg or 8 µg of Megamer donor
– Plate cells in 12-well plate
Day 5:
– Flow cytometry:
Total editing: GFP-
HDR: mCherry+
~100 nt ~100 nt

28. 28
0
10
20
30
40
50
60
70
80
90
100
0
2
4
6
8
10
12
14
16
18
20
4ug 8ug
Megamer ssDNA
(sense strand)
no donor Cell only
Totalediting(%)
HDR(%)
HDR Total Editing
Efficient insertion of mCherry using Cas9 D10A and Megamer

29. HDR donor design when using Cas9 D10A nickase
• Point mutations and small (<60 nt) insertions or tags
– ssODNs (Ultramer DNA Oligonucleotides)
• Place intended insert in between nick sites
• 40 nt homology arms beyond the nick sites
• Modifications (Phosphorothioate Bonds, etc.) at ends can increase exonuclease resistance
• Test both top and bottom strands, if possible
• Large corrections and insertion of new coding material
– Longer ssDNAs (Megamer ssDNA Fragments)
• Place intended insert in between nick sites
• 100 nt homology arms beyond the nick sites
• Test both top and bottom strands, if possible
29

30. Outline: Using Alt-R Cas9 nickases for genome editing
• Background: Genome editing using the Alt-R CRISPR-Cas9 system
• Introduction of Alt-R Cas9 D10A and Cas9 H840A Nickases
• Using Cas9 nickases for genome editing
- How to design gRNA pair
- How to design donor template for homology directed repair (HDR)
• Workflow: Doing genome editing with Cas9 nickases
30

31. CRISPR analytical process—nickase HDR experiment
31

32. Case study: insertion at AAVS1 safe harbor site
• Goal: insert 6 bp XhoI site (CTCGAG) at the indicated position in the
AAVS1 safe harbor site
32
AAVS1 genomic DNA sequence:
CCAATCCTGTCCCTAGTGGCCCCACTGTGGGGTGG
AGGGGACAGATA|AAAGTACCCAGAACCAGAGCCA
CATTAACCGGCCCTGGGAATATAAGG
Desired AAVS1 genomic DNA sequence:
CCAATCCTGTCCCTAGTGGCCCCACTGTGGGGTGG
AGGGGACAGATACTCGAGAAAGTACCCAGAACCAG
AGCCACATTAACCGGCCCTGGGAATATAAGG

33. IDT CRISPR gRNA design tool
33

34. Output from the gRNA design tool
34

35. Closest Cas9 cleavage sites are ≥12 nt from desired
insertion site
35
13 nt 12 nt

36. HDR rate decreases significantly at positions >10 bases
away from CRISPR-Cas9 cut site
36
0
5
10
15
20
25
30
no
ssODN
-27 -21 -15 -10 -6 -4 -2 0 2 4 6 10 15 21 27
EcoRI cleavage (%)
Insertion position away from cut site (base pairs)

37. Closest guides give low total editing and HDR frequency
with WT Cas9
0
20
40
60
80
100
Top Btm Top Btm
WT Left 13 nt WT Right 12 nt
Cleavage (%)
Wild type Cas9 targeting AAVS1
HEK-293 cells, Nucleofection
HDR Total editing
37

38. Test HDR efficiency using Cas9 D10A with recommended
gRNA pair design considerations
38
20 nt 26 nt
PAM-out
46 nt
between
nicks

39. Cas9 D10A nickase enables HDR at a site lacking efficient
WT Cas9 guide
39
0
20
40
60
80
100
Top Btm Top Btm Top Btm
WT Left 13 nt WT Right 12 nt D10A 46 nt PAM-out
Cleavage (%)
Wild type vs D10A nickase targeting AAVS1
HEK-293 cells, Nucleofection
HDR Total editing

40. Conclusions
• Use gRNA pair in PAM-out orientation for both D10A and H840A
• Make sure gRNA pair is at an optimal distance
Ø D10A: targeted nick sites separated by 37–68 bp
Ø H840A: targeted nick sites separated by 51–68 bp
• D10A is more potent than H840A at mediating HDR
• HDR design rules when using D10A Nickase:
– Place intended insert in between nick sites
– Small insertions and tags: 40 nt homology arms
– Larger insertions: 100 nt homology arms
– Test both top and bottom strand, if possible
• Cas9 D10A nickase enables HDR at sites lacking efficient WT Cas9
guide
40

41. THANK YOU
41

42. Take home messages
• PAM-out orientation
– D10A: 40–70 bp between nicks
– H840A: 50–70 bp between nicks
• D10A > H840A for HDR
• D10A > WT for sites without a
nearby guide
42
More information and protocols available at: www.idtdna.com/crispr-cas9
(Go to the Resources section for protocols)
More webinars at: youtube.com/idtdnabio