reducing off-target events in crispr genome editing
TRANSCRIPT
Reducing off-target events in CRISPR genome editing applications with a novel, high-fidelity
Cas9 nucleaseChristopher Vakulskas, PhD
Staff Scientist
1
Outline: Alt-R® S.p. HiFi Cas9 Nuclease 3NLS
• Background• CRISPR delivery mechanism matters
– Constitutive vs. transient Cas9 expression• Existing high-fidelity Cas9 mutants perform poorly with ribonucleoprotein (RNP)
– Reduced off-target editing (OTE) at the expense of on-target potency• Alt-R S.p. HiFi Cas9 development
– On-target performance– Off-target performance
• Alt-R S.p. HiFi Cas9 applications– Editing in CD34+ hematopoietic stem/progenitor cells (HSPCs)– Editing in mouse cells– Homology-directed repair
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Genome editing
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CRISPR-Cas9 genome editing
• RNA-guided endonuclease• PAM site (NGG)• crRNA and tracrRNA• Blunt-ended cut sites
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Cas9 sgRNA vs. crRNA:tracrRNA complexes
crRNA:tracrRNA complex• Not ideal to express from DNA• Not ideal for IVT• Efficient for chemical synthesis
– 20 bases unique, 16 bases constant– 67 bases universal tracrRNA
• Easy to modify, escape immune response
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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, IVTs cannot be modified
Transfection of IVT sgRNAs can be toxic to cells
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• Successful gene editing• Transfection of IVT sgRNAs sometimes result in:
– Large scale cell death– Induction of innate immune response
HEK-293 cells only 30 nM sgRNA IVT 30 nM 2-part RNA
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
Implementing CRISPR-Cas9 gene editing
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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 nMElectroporation: 1–3 µM
Microinjection
Cas9
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On-target site
Empirically determinedoff-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)
Ratio of on/off target editing depends heavily on Cas9 source(i.e., plasmid, mRNA, or protein)
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Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol, 208:44–53.
RNP delivery of wild-type Cas9 reduces off-target editing
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GAGTCCGAGCAGAAGAAGAAGGG EMX1 on-target siteGAGTTAGAGCAGAAGAAGAAAGG Off-target site 1GAGTCTAAGCAGAAGAAGAAGAG 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
Inde
l by
NG
S (%
)
On targetOff target 1Off target 2
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)
NGS analysis of “cell-free” Cas9 cleavage sites
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0
50
100
150
200
250
300
350
Cle
avag
e fre
quen
cy (N
GS
read
s)
On target Off target SVM-Predicted off-target sites
Predicted Not predicted
9%
AR-S-1893GTTGGAGCATCTGAGTCCAGGGG
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?
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Published, rationally-designed, high-fidelity Cas9 mutants
• eSpCas9– 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.
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Evaluation of RNPs generated using high-fidelity vs. wild-type Cas9 enzymes• We introduced the eSpCas9 and SpCas9-HF1 point mutations into
the Alt-R S.p. Cas9 3NLS purification plasmid• WT, SpCas9-HF1, and eSpCas9 were purified to homogeneity • All mutant proteins were compared to WT for on-target performance
– Multiple crRNAs at multiple genomic loci– Delivered into HEK-293 cells by Lipofection, 10 nM dose
• T7EI assay used to evaluate editing efficiency after 48 hr
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Slaymaker IM, Gao L, et al. (2016) Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268):84–88.
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.
eSpCas9 and SpCas9-HF1: On-target performance is significantly compromised
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0
20
40
60
80
100
HBB CCR5 HEXB TRAC
Inde
l by
NG
S (%
)
WTeSpCas9SpCas9-HF1
0102030405060708090
T7EI
cle
avag
e (%
) WTeSpCas9SpCas9-HFI
Alt-R CRISPR-Cas9 System10 nM RNP, LipofectionHPRT locusHEK-293 cells
Chemically modified sgRNAs Amaxa® Nucleofector® (Lonza)CD34+ HSPCs
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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Bacterial screen to identify novel high-fidelity Cas9 mutants
• Double selection for avoidance of off-target cleavage and maintenance of on-target cleavage
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On- and off-target performance of novel mutations
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HEKsite4
0
2
4
6
8
10
12
14
16
WT 85 82 79 76 73 70 67 64 61 58 55 52 49 46 43 40 37 34 31 28 25 22 19 16 13 10 7 4 1
Dis
crim
inat
ion
ratio
(on/
off t
arge
t edi
ting)
Low OTE mutants (rank ordered by performance)
Evaluating mutant Cas9 performance in cultured cells• All experiments were performed with Alt-R CRISPR-Cas9 System
components, using RNP editing protocol• Conditions tested:
– Lipofection• 10 nM RNP complexed with Lipofectamine® RNAiMAX reagent (Thermo Fisher)• HEK-293 cells• DNA isolated 48 hr post transfection
– Electroporation• Typically 3 mM RNP delivered with the Amaxa Nucleofector (Lonza) instrument• HEK-293 cells• DNA isolated 48 hr post transfection
• Alt-R Genome Editing Detection Kit• NGS analysis
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Alt-R S.p. HiFi Cas9 Nuclease 3NLS: On-target performance is significantly improved
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Alt-R CRISPR-Cas9 System10 nM RNP, Lipofection HPRT locusHEK-293 cells
Chemically modified sgRNAs Amaxa® Nucleofector® (Lonza)CD34+ HSPCs
0
20
40
60
80
100
HBB CCR5 HEXB TRAC
Inde
l by
NG
S (%
)
WT Cas9eSpCas9SpCas9-HF1Alt-R HiFi Cas9
0102030405060708090
T7EI
cle
avag
e (%
)
WT Cas9eSpCas9SpCas9-HFIAlt-R HiFi Cas9
Danny DeverMatt PorteusStanford University
Alt-R S.p. HiFi Cas9 Nuclease 3NLS reduces off-target editing while maintaining on-target potency
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Published, known off target sites
0
10
20
30
40
50
60
On-target Off-target On-target Off-target On-target Off-target
EMX1 HEKSite4 VEGFA3
T7EI
cle
avag
e (%
)
WT Cas9eSpCas9SpCas9-HFIAlt-R HiFi Cas9
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 developed a proprietary method to analyze global off-target editing in a cell-free Cas9 cleavage system
• The off-target editing percentage should be an overestimation– Cas9 RNP should be stable in the absence of cellular proteases and
nucleases
– Purified genomic DNA is absent factors that would encumber Cas9 binding
• We compared WT and Alt-R HiFi Cas9 total editing using 5 unique crRNAs that target different genomic loci
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WT Alt-R S.p. Cas9
Alt-R S.p. HiFi Cas9
EMX1AR METGRHPRCTNNB1
Global and unbiased analysis of OTE: WT vs. HiFi
On-target editing Off-target editing
85%
15%
85%
15%
81%
19%
72%
18%
71%
29%
90%
10%
100%
90%
10%
52%48% 100%
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Global and unbiased analysis of OTE: WT vs. HiFi
27*On-target Cleavage Site
0
50
100
150
200
250
300
350
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71
Cas
9 cl
eava
ge (c
ount
s)
Genomic loci (Rank order: Most to least frequently cleaved by WT Cas9)
15%
85%
WT Cas9
52% 48%
Alt-R HiFi Cas9
% Off-target cleavage% On-target cleavage
Alt-R S.p. HiFi Cas9Alt-R S.p. WT Cas9
* AR-S-1893GTTGGAGCATCTGAGTCCAGGGG
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.
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On-target site
Empirically determinedoff-target sites
S.p. Cas9 is likely to produce off-target cleavage(particularly with plasmid expressed sgRNA and Cas9)
RNP dose response: WT Cas9 vs. Alt-R HiFi Cas9
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0.1
1
10
100
4 µM 2 µM 1 µM 0.5 µM 4 µM 2 µM 1 µM 0.5 µM
WT Alt-R S.p. Cas9 Alt-R S.p HiFi Cas9
Inde
l by
NG
S (%
; log
10)
On target
Off target 1
Off target 2
Ex-vivo genome editing will be an early medical application
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Alt-R S.p. HiFi Cas9 Nuclease 3NLS in human, CD34+ HSPCs at the HBB locus
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Danny DeverMatt PorteusStanford University
0
20
40
60
80
100
120
HBB CCR5 HEXB TRAC
Inde
l(%
; nor
mal
ized
to W
T)
WT Cas9Alt-R HiFi Cas9eSpCas9SpCas9-HF1
Alt-R S.p. HiFi Cas9 allows specific and efficient HDR
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Danny DeverMatt PorteusStanford University
0
20
40
60
80
100
120
HBB CCR5 HEXB TRAC
Targ
eted
CD
34+
HSP
Cs
(%; n
orm
aliz
ed to
WT)
WT Cas9Alt-R HiFi Cas9eSpCas9SpCas9-HF1
Alt-R S.p. HiFi Cas9 retains high on-target potency in mouse cells (Hepa1-6)
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0
10
20
30
40
50
60
70
80
90T7EICleavage(%
)Alt-R S.p. HiFi Cas9WT Cas9
Conclusions
• Off-target effects are a very real concern when using CRISPR methods• Using DNA-free RNP methods inherently gives lower OTEs• Cas9 mutations that improve off-target profile can also significantly hurt
on-target cleavage, especially when using preferred RNP methods• New IDT HiFi Cas9 Nuclease 3NLS retains on-target cleavage and has
reduced off-target risk• There is no way to ensure 100% on-target, 0% off-target, but you can
minimize risk by using RNP, careful selection of target site, and use of mutant higher specificity enzymes
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Alt-R HiFi Cas9 RNP mitigates off target risk
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1
10
100
On target Off target 1 Off target 2
Inde
l by
NG
S (%
; log
10)
HEK-WT Cas9 CellsWT Cas9 RNP (2 µM)HiFi Cas9 RNP (2 µM)
THANK YOU
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