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Ashley Jacobi, Research ScientistIntegrated DNA Technologies
New RNA tools for optimized CRISPR/Cas9 genome editing
October 7th, 2015
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Implementing CRISPR/Cas9 gene editing
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Options for the CRISPR gRNA
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Repair of double-stranded breaks—HR vs. NHEJ
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• S. pyogenes Cas9 is a large protein, 1368 aa = 4104 bp
• Plasmid containing Cas9: 7–10 kb
• Transfection of a large plasmid results in variable and low transfection efficiency, making large quantitative comparison studies difficult
Delivery of a Cas9 + sgRNA expression plasmid is difficult
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Delivering large Cas9 expression plasmid to cells can be difficult
Optimizing CRISPR gRNA using HEK293-Cas9 cell line
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Low, constant level of Cas9 present in HEK293-Cas9– Note the extremely high levels of Cas9 present in
just a small fraction (~10%) of transfected cells using plasmid. Can this contribute to OTEs?HEK293-‐Cas9 Cells
Western blot—Cas9 primary antibody
T7EI mismatch detection to assay gene disruption1. Transfect HEK-Cas9 cells with the CRISPR gRNA
– Alternatively deliver Cas9 as plasmid, mRNA or protein
2. Incubate 48 hours, then harvest genomic DNA3. PCR amplify region around CRISPR site (400–1000 base amplicons)
– Heat, cool to form heteroduplexes
4. Incubate with T7 Endonuclease I (T7EI, New England BioLabs) 5. Run on gel or Fragment Analyzer™ (Advanced Analytical) to visualize cleavage at heteroduplex mismatch sites
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IDT CRISPR gene editing and mutation detection workflow
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• The Fragment Analyzer™ (Advanced Analytical) provides reliable quantification of T7EI heteroduplex cleavage assay with 96-channel CE– High resolution analysis of fragments 10–40,000 bp– Rapid 1 hr run – 1/10th amount of DNA required to visualize
Transfect 2-‐part RNA at 30 nM or gBlocks fragment at 3 nM into Cas9 expressing cells
Extract gDNA after 48 hr with
QuickExtract DNA Solution
Heat gDNA extract at 65°C for 15 min followed by 95°C
for 15 min
Amplify gDNA with KAPA HiFi
Polymerase and PCR assay targeting region of interest
Add NEB buffer 2 to PCR, heat to 95°C and slowly cool to allow heteroduplex
formation
Digest heteroduplexes
with 2 units of T7EI at 37°C for 1 hr
Analyze digestion on Fragment Analyzer
Electropherogramand peak table of separated sample on Fragment Analyzer
52% T7 cleavage
Options for the CRISPR gRNA
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gBlocks® Gene Fragments for CRISPR• Inexpensive gene synthesis product with rapid delivery
– High quality double-stranded DNA fragments– 125–2000 bp in length– Sequence verified
• CRISPR gBlocks® Gene Fragment = 364 bp sgRNA expression cassette – Comprised of a 265 bp U6 promoter that drives transcription of a 99 base sgRNA
www.idtdna.com/CRISPR
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AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT
gBlocks® Gene Fragments as sgRNA (three methods)
1. Clone gBlocks Gene Fragments into an expression plasmid
2. Use gBlocks Gene Fragment as template for in vitro transcribed sgRNA (IVT sgRNA)
3. Directly transfect gBlocks Gene Fragment into cells without cloning
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www.idtdna.com/CRISPR
http://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf
gRNAbackbone
Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.
>14,000 gBlocks® Gene Fragments manufactured for CRISPR
CRISPR gBlocks® Gene Fragment in HPRT gene (HEK293 Cas9 cells)
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38094S
38095S
38115S
38129S
38231S
38239S
38256S
38338S
38371S
38448S
38478S
38509S
38510S
38574S
38626S
+
23% 46% 21% 0% 31% 27% 3% 47% 0% 41% 14% 39% 4% 36%43%
2% Agarose gel
Fragment Analyzer™
Note: sequence analysis shows 30% cleavage in T7EI assay = 60–70% total editing
+
Validation of T7EI assay: % total editing compared to Sanger sequencing
Sanger sequence analysis shows 30% cleavage in the T7EI assay = 60–70% actual change at DNA level (T7EI misses small changes like single base indels)
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• Amplicons resulting in varying editing efficiencies via T7EI were cloned and sequenced.
T7EI cleavage (%)
CRISPR gBlocks® Gene Fragments sgRNA perform well across many sites(3 genes; 301 sites)
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0102030405060708090100
% Cleavage via 2U
T7EI
167 EMX1 Exon 3 (64% GC)
91%*
0102030405060708090100
% Cleavage via 2U
T7E1I
92 STAT3 Exon 5/6 (47% GC)
76%*
0102030405060708090100
% Cleavage via 2U
T7EI
42 HPRT Exon 7 (36% GC)
81%*
sgRNAs expressed from gBlocks Gene Fragments work well without the need to clone into plasmids
Directly transfect into HEK-Cas9 cells at 3 nMEvery PAM site in 3 exons
* Percentage of sgRNA designs with >20% editing efficiency by T7EI assay
+
Options for the CRISPR gRNA
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Options for the CRISPR gRNA
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UUAUAUCCAACACUUCGUGGUUUUAGA-‐-‐GCUAG||||||| |||| A||||||| |||| A
C-‐GGAAUAAAAUUGAACGAUAU| ||A| ||GUCCGUUAUCAACUUG
|||| A|||| A
AGCCACGGUGAAAG ||||||UCGGUGCUUU
sgRNA: 99–123 bases (99mer shown)
20 base “protospacer” guide, target specific
• Near the length limit for chemical manufacturing• Expensive to chemically make long sgRNAs for many sitesThis form is used in our gBlocks® Gene Fragment sgRNAexpression cassette
UUAUAUCCAACACUUCGUGGUUUUAGA-‐-‐GCUAUGCUGUUUUG||||||| ||||||||||||||
C-‐GGAAUAAAAUUGAACGAUACGACAAAACUUACCAAGGUUGU| ||A| ||GUCCGUUAUCAACUUG
|||| A|||| A
AGCCACGGUGAAAG ||||||UCGGUGCUUUUUUU
crRNA: 42 bases (target specific)tracrRNA: 89 bases (universal)
• 42mer target specific (20 base target, 22 base constant)• 89mer universal tracrRNA—can be made in bulk, making
them more affordableCan these be optimized and shortened to improve function and lower cost?
Optimized length of crRNA and tracrRNA
crRNA: 42 bases (20+22)tracrRNA: 89 bases (universal)
UUAUAUCCAACACUUCGUGGUUUUAGA-‐-‐GCUAUGCUGUUUUG||||||| ||||||||||||||
C-‐GGAAUAAAAUUGAACGAUACGACAAAACUUACCAAGGUUGU| ||A| ||GUCCGUUAUCAACUUG
|||| A|||| A
AGCCACGGUGAAAG ||||||UCGGUGCUUUUUUU
Native sequence
Shorter
Shorter…
Short
Both the crRNA and the tracrRNA can be truncated
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1. Short/Short
2. Long/Short
3. Short/Long
4. Long/Long Worse
Worse when too short
Better
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0
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20
30
40
50
60
70
80
90
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89 nt tracrRNA 74 nt tracrRNA 70 nt tracrRNA 67 nt tracrRNA 65 nt tracrRNA 63 nt tracrRNA
T7EI cleavage (%
)
42-‐nt crRNA39-‐nt crRNA36-‐nt crRNA34-‐nt crRNA
Length optimization of crRNA & tracrRNAHPRT 38285 gRNA (HEK293 Cas9 Cells)
Optimal length for crRNA is 36 nt; optimal tracrRNA is 67 nt
+
0
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20
30
40
50
60
70
80
90
100
HPRT 38094 S
HPRT 38231 S
HPRT 38371 S
HPRT 38509 S
HPRT 38574 S
HPRT 38087 AS
HPRT 38133 AS
HPRT 38285 AS
HPRT 38287 AS
HPRT 38358 AS
HPRT 38636 AS
HPRT 38673 AS
T7EI cleavage (%
)
CRISPR gRNA Comparison—12 gRNAs Targeting HPRT(HEK293-‐Cas9 Cells)
2-‐part RNA (36/67)Native RNA (42/89)In vitro transcribed sgRNAsgRNA Expression Plasmid (2.7 kb)gBlocks Gene Fragments sgRNA
Optimized 2-part CRISPR RNAs are superior to other gRNAs
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HEK-‐Cas9 cells
2-‐part RNA (30 nM)IVT RNA (30 nM)Plasmid (100 ng)
gBlocks Fragment (3 nM)
+ or or or
Highly purified oligos are necessary for longer tracrRNAs
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HEK-‐Cas9 cells
2-‐part RNA, 30 nM
Desalt crRNA
Desalt vs. HPLC tracrRNA
1. Shortened crRNA:tracrRNA performs better than native form2. 67mer tracrRNA functions well as desalted, shows slight improvement as HPLC3. Native 89mer tracrRNA requires highly purified synthesis
0
10
20
30
40
50
60
70
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HPRT 38094 S
HPRT 38231 S
HPRT 38371 S
HPRT 38509 S
HPRT 38574 S
HPRT 38087 AS
HPRT 38133 AS
HPRT 38285 AS
HPRT 38287 AS
HPRT 38358 AS
HPRT 38636 AS
HPRT 38673 AS
T7EI cleavage (%)
HPLC vs. Desalted RNA Oligos – HPRT 12 sites(HEK293 Cas9 cells)
short crRNA (36nt) : short tracrRNA desalt (67nt)
short crRNA (36nt) : short tracrRNA HPLC (67nt)
long crRNA (42nt) : long tracrRNA desalt (89nt)
long crRNA (42nt) : long tracrRNA HPLC (89nt)
+
2-part RNA oligos will be available from IDT this month!• crRNA
– 36 nt custom desalted RNA oligo– Ability to order in 96-well plate format– 2 or 10 nmol
• tracrRNA– 67 nt HPLC purified RNA oligo– Modified for nuclease stability– 5, 20, or 100 nmol
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Be sure to check QC data on long synthetic RNAs
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IDT tracrRNA Vendor “X” tracrRNA
ESI-‐MS
2-part RNA system functions well across many sites
24All PAM sites in 6 exons, 553 sites (HEK293 Cas9 Cells)
* **
* * *
* Percentage of sgRNA designs with >20% editing efficiency by T7EI assay+
Summary of 2-part RNA functional performance in HEK-Cas9 cells
553 guide sites in 6 exons from 4 human genes
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Target amplicon Number of sites % >15% T7 cleavage % >20% T7 cleavage
HPRT exon 7 (36% GC) 42 40/42 = 95% 40/42 = 95%
HPRT exon 1 (62% GC) 164 135/164 = 82% 123/164 = 75%
EMX1 exon 3 (64% GC) 167 151/167 = 90% 143/167 = 86%
EMX1 exon 5/6 (40% GC) 59 56/59 = 95% 53/59 = 90%
STAT3 exon 5/6 (47% GC) 92 86/92 = 93% 86/92 = 93%
DICER exon 8 (38% GC) 29 28/29 = 97% 28/29 = 97%
Total 553 496/553 = 90% 473/553 = 86%
+
Comparison of gBlocks® Gene Fragments sgRNAs vs. 2-part RNA
26+ or
**
**
**
CRISPR on-target mutation profiles for varying RNA Triggers
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• Sanger sequencing of amplicons from one target site in HPRT as varying RNA triggers
Although use of shorter 17mer guides has been reported to reduce off-target effects, 17mer guides can result in far worse on-target results.
Other design features to consider — shorter protospacer?
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0102030405060708090100
HPRT 38094 S
HPRT 38231 S
HPRT 38371 S
HPRT 38509 S
HPRT 38574 S
HPRT 38087 AS
HPRT 38133 AS
HPRT 38285 AS
HPRT 38287 AS
HPRT 38358 AS
HPRT 38636 AS
HPRT 38673 AS
T7EI cleavage (%
)
Guide RNA length study—20 vs. 19 vs. 18 vs. 17 nt(HEK293 Cas9 Cells)
20 nt 19 nt 18 nt 17 nt
+
• Transfection of in vitro transcribed sgRNAs sometimes resulted in large scale cell death.
Comparison of in vitro transcribed sgRNAs to 2-part RNAs
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MEGAshortscript T7 IVT Kit (Ambion)
HiScribe T7 High Yield IVT Kit (NEB)
Activity of 2-part RNAs vs. in vitro transcribed sgRNAs
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0
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20
30
40
50
60
70
80
90
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HPRT 38094 S
HPRT 38231 S
HPRT 38371 S
HPRT 38509 S
HPRT 38574 S
HPRT 38087 AS
HPRT 38133 AS
HPRT 38285 AS
HPRT 38287 AS
HPRT 38358 AS
HPRT 38636 AS
HPRT 38673 AS
T7EI cleavage (%)
2-‐part RNA vs. IVT sgRNA—HPRT 12 sites(HEK293 Cas9 Cells)
2 part RNA
IVT sgRNA-‐
+ or
In vitro transcribed sgRNAs trigger immune response, 2-part RNA oligos do not (HEK-Cas9 cells)
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• IFITM1, RIGI, and OAS2 had similarly high induction when treated with in vitro transcribed sgRNA (triphosphate removed)
• No inductions were detected when treated with 2-part RNA oligos
+ or
• Reverse transfection into 96-well plate• 100 ng plasmid, 0.3 µL TransIT X2• Images taken 48 hr after transfection
Back to the Cas9 delivery problem…
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IDT Cas9 Expression Plasmid—minimal vector
• Minimal vector (7.3 kb)– Origin of replication– Ampicillin resistance– No selection marker
• Deliver Cas9 expression plasmid, followed by delivery of 2-part RNA
• Improvements in editing, other plasmid associated problems remain
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• Simple, fast, and robust delivery– Complex gRNA & Cas9 protein– Deliver directly to cells using lipofection or
electroporation
• Cas9 RNP = preferred method– Protection of RNA—reduced risk of degradation– Higher editing compared to plasmid delivery– No DNA present—no integration events– Tight control of Cas9 (on/off, nothing present in the cell
that can make more)– Reduced risk of mosaicism in animal embryo studies
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Delivery of CRISPR gRNA + Cas9 protein as ribonucleoprotein complex (RNP)
+
2 part gRNA delivered into HEK-Cas9 cells and RNP delivered into normal HEK cells result in identical editing efficiency
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0
10
20
30
40
50
60
70
80
90
100
HPRT 38094 S
HPRT 38231 S
HPRT 38371 S
HPRT 38509 S
HPRT 38574 S
HPRT 38087 AS
HPRT 38133 AS
HPRT 38285 AS
HPRT 38287 AS
HPRT 38358 AS
HPRT 38636 AS
HPRT 38673 AS
T7EI cleavage (%
)
HPRT 12 Sites -‐ 2 part RNA (30 nM) into HEK293-‐Cas9 Cells – 0.75 µL RNAiMAX2 part RNA + Cas9 protein into HEK293 Cells
10 nM gRNA, 10 nM Cas9 protein –1.2 µL RNAiMAX
HEK293-‐Cas9 Cells HEK293 cells + Ribonucleoprotein Complex
+or
2 part + Cas9 protein RNP Delivery is efficient in many cell lines
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0
10
20
30
40
50
60
70
80
90
100
Mm F9 gRNA-‐1
Mm F9 gRNA-‐2
Mm F9 gRNA-‐3
Mm F9 gRNA-‐4
Mm F9 gRNA-‐5
Mm F9 gRNA-‐6
Mm F9 gRNA-‐7
Mm F9 gRNA-‐8
Mm F9 gRNA-‐9
Mm F9 gRNA-‐10
T7EI cleavage (%
)
Mm Factor IX gRNA Screen – AML12 CellsCas9 RNP—10nM gRNA, 10nM Cas9 protein, 1.25µL RNAiMAX
9/10 sites high % gene editing
+
Conclusions
1. Synthetic RNA oligos mimicking the natural 2-part CRISPR system (crRNA:tracrRNA complex) function well in mammalian cells, both when Cas9 is expressed in the target cell and when pre-complexed with Cas9 protein as a ribonucleoprotein.
2. “Optimized” shortened crRNA:tracrRNA complex shows improved editing activity.
3. In vitro transcribed sgRNAs have a risk for immune activation.
4. The 2-part system & gBlocks® Gene Fragments both give a high positive hit rate in gene walks. This suggests that site selection algorithms may not be needed for many applications, as long as multiple sites are tested.
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Coming soon: New CRISPR products from IDT!
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CRISPR product type
CRISPR crRNA 2 and 10 nmol
CRISPR crRNA plates 2 nmol
CRISPR tracrRNA 5, 20, and 100 nmol
Control kits Human, mouse, and rat
CRISPR crRNA HPRT positive control Human, mouse, and rat
CRISPR crRNA negative controls 3 sequence options
HPRT PCR primer mix Human, mouse, and rat
Cas9 expression plasmid
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