increasing genome editing efficiency and specificity with...
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Increasing genome editing efficiency and specificity
with optimized CRISPR-Cas9 guide RNAs
Ashley Jacobi
Senior Staff Scientist – Molecular Genetics
1
Outline
• Background – CRISPR genome editing
• Developments and improvements of guide RNAs (gRNAs)
– In vitro transcribed single guide RNA (sgRNA)
– Chemically synthesized 2-part crRNA + tracrRNA
– Chemically synthesized sgRNA
– Benefits of introducing chemical modifications
– Which gRNA to use and when?
• Optimal delivery with different sources of Cas9
• Off-target analysis of different gRNA and Cas9 formats
• Brief introduction of newest Alt-R CRISPR reagent
• 2
Common CRISPR RNA-guided Restriction Endonucleases
3
AsCas12a (Cpf1)SpCas9
“NGG” PAM site
dsDNA cut with blunt ends
Separate crRNA + tracrRNA (annealed)
20 nt protospacer
“TTTV” PAM site
5 base staggered cuts
Single, short crRNA
21-24 nt protospacer
Implementing CRISPR-Cas9 gene editing
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(RNP)
Alt-R CRISPR-Cas9 RNP System—fast, highly efficient deliveryRNP delivery with 2-part guide RNA (crRNA + tracrRNA)
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Alt-R CRISPR-Cas9 RNP System—fast, highly efficient deliveryRNP delivery with sgRNA
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Chemically synthesized guide RNAs
Developments and improvements
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Transfection of IVT sgRNAs can be toxic to cells
8
• 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 IVT sgRNA 30 nM 2-part gRNA
IFNa stimulation in human PBMCs using unmodified gRNAs
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PBMC = peripheral blood mononuclear cell
Schubert et al. (2018) J Cytokine Biol 3:1
No IFN response with modified,
chemically synthesized Alt-R
gRNAs
Benefits of chemically synthesized gRNAs
• Chemical modifications can be added
– Improve nuclease stability
– Increase efficiency
– Reduce risk of triggering innate immune response
• Experiment-ready reagents are easily used
– crRNAs (36 nt) have the target specific sequence, can be synthesized by the
thousands in small scale, can be provided as libraries
– tracrRNAs (67 nt) has the universal sequence, available in larger scales, anneal to
each crRNA
– sgRNAs (full 100 nt) do not require annealing
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Empirically determined tolerance to chemical
modification in crRNA and tracrRNA regions
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5’ C*U*U*AUAUCCAACACuuCGuGGuuuUAGAGCUAU*G*C*U 3’
16-base tracrRNA
binding domain20-base protospacer
guide domain
crRNA
cGGAAuAAaAuuGAACGAUAC*G*A 5’
u| ||
A| ||
gucCguuAUCAACUUG
|||| A
|||| A
AGCCACGGUGAAA
G ||||||
UCGGUGCU*U*U 3’
tracrRNA
AGCU = 2’OMe RNA
agcu = RNA
“*” = PS bond
= Major loss of function with 2’-mod
= Minor loss of function with 2’-mod
= Loss of function varies with sequence
Alt-R gRNA options—crRNA + tracrRNA & sgRNA
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Alt-R
2 part
Alt-R
2 part XT
Alt-R
sgRNAStructure
gRNA format Alt-R CRISPR-Cas9
crRNA & tracrRNA
Alt-R CRISPR-Cas9
crRNA XT & tracrRNA
Alt-R CRISPR-Cas9
sgRNA
ComponentscrRNA
tracrRNA
crRNA XT
tracrRNAsgRNA
Sizes (nt)36
67
36
67 100
Annealing
requiredYes Yes No
Modifications Moderate High Moderate
Applications • Cas9-expressing cells
• RNP in most cell types
• Co-delivery with Cas9 plasmid/Cas9 mRNA
• RNP under difficult experimental conditions
(e.g., high nuclease environments)
All options have a 3–5 day turnaround time
DNA/mRNA Cas9 expression vectors can be co-delivered successfully with
more highly modified gRNA variants (HEK-293 cells)
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12 sites targeting HPRT
Amaxa® Nucleofection® (Lonza) in HEK-293 cells
RNP delivery
• gRNA:Cas9 protein = 1.2:1 ratio
• 3 µM Alt-R Electroporation Enhancer
Cas9 plasmid & mRNA
• Co-delivery with gRNA
• 1 µg of plasmid or mRNA
Indel (insertions/deletions) by NGS
• All gRNA forms work equally
well with RNP delivery
DNA/mRNA Cas9 expression vectors can be co-delivered successfully
with more highly modified gRNA variants (HEK-293 & K562 cells)
14
12 sites targeting HPRT
Amaxa Nucleofection in HEK-293 & K562 cells
RNP delivery
• gRNA:Cas9 protein = 1.2:1 ratio
• 3 µM Alt-R Electroporation enhancer
Cas9 plasmid & mRNA
• Co-delivery with gRNA
• 1 µg of plasmid or mRNA
Indel by NGS
• All gRNA forms work equally
well with RNP delivery
Efficiency comparison of gRNA forms in CD34+ cells
• Collaboration with Ayal Hendel Lab (Bar-Ilan University)
• Goal: to efficiently knock-out genes associated with severe combined immunodeficiencies; identify the best gRNA forms to do so with RNP delivery
• Method: Electroporation into CD34+ HSPCs
• Alt-R Cas9 Nuclease V3
• In vitro transcribed sgRNA, Alt-R crRNA:tracrRNA, Alt-R crRNA XT:tracrRNA, Alt-R sgRNA
• Alt-R electroporation enhancer (with or without)
– 100 nt ssDNA
– Non-homologous to human, mouse and rat genomes
– Increases electroporation efficiency of RNP complexes
Titration of RNP complexes
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High editing in CD34+ cells using Alt-R RNP complexesRAG2 gRNA variants have similar activity
Alt-R electroporation enhancer significantly boosts editing
levels for all gRNA forms in CD34+ cells.
High editing in CD34+ cells using Alt-R RNP complexesHighly modified gRNAs are more efficient for RAG1
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RNP concentration (µM)
Alt-R electroporation enhancer significantly boosts editing
levels for all gRNA forms in CD34+ cells.
Off-target analysis of different gRNA and Cas9 formats
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CRISPR-Cas9 off-target effects (OTEs)
• Delivering Cas9 in an expression vector increases non-specific
editing due to continued and long-lasting Cas9 synthesis
• 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
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Bacterial screen to identify novel high-fidelity Cas9 mutants
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22
Nature Medicine (2018) 24:1216–1224
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New IDT HiFi Cas9 mutant retains on-target activity &
increases specificityAlt-R
HiFiCas9
On- & off-target comparison of different gRNA forms
24
Sequence Information:
CRISPR-Cas9 off-target detection
• Methods to predict or validate off-target sites
– In silico predictive tools
• Difficult, often miss important sites, over-prediction
– In vitro assays to define possible OTEs
• SITE-seq, CIRCLE-seq, etc.
• Often over predictive, waste sequencing read space
– GUIDEseq identification of OTEs
• Cell line of interest; mostly quantitative
– rhAmpSeq system for CRISPR: a multiplexed, amplification-based, target
enrichment next-generation sequencing approach
• Quantitative assessment of on-target site and up to 1000 off-target sites in a single
multiplexed reaction
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Identification of potential off-target sites for AR & EMX1
• GUIDEseq into Cas9 cells,
10 µM Alt-R crRNA XT:tracrRNA
• In silico predictions
• rhAmpSeq assay panel designed
and synthesized
• AR panel = 54 assays
• EMX1 panel = 32 assays
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Strand A A T T A T G G G G A T T A C T A G G A GS Reads
- A A T T A T G G G G A T T A C T A G G A 4248
+ C A T T A T G G G G A T T - C T A G G A 359
- T A T T A A G G G A A C T A C T A G G A 328
- G A T G A T G G G G A T T T C T A G G A 136
- T A T T A A G G G G A T T A C T A G G G 121
+ A T T A G A G G G G A C T A C T A G G G 62
+ T T T T A T G G G G A G T A - T A G G A 49
+ T G T T A T A G G G A C C A C T A G G A 43
+ A A T A A T G G G G A T T A C T A G G A 40
+ G A C A C T G G G G A T T A C T A G G A 33
- T G T T A T A G G G A C C A C T A G G A 20
- A T T T A C A G G G A T T A C T T G G T 15
+ T A G T A T G G G A A T T T C T A G G A 15
+ A A T G A T G G G A A C T A C T A G A A 14
- T A T T A T G G G G A A T A C T A G G A 11
- C T T T A T G G G G A T T A C T A T G A 11
+ A A C T A T G G T G A C T A C T A G G A 9
+ A C T T T G G G G G A T T G C T A G G A 8
+ T T G A A T G G G G A T T A C T T G G A 8
+ C A A G T T A G G G A T T A C T A G G A 6
- T A C T A T G G G G A T T A C T A G G A 4
- T G G T A T G G G G G A T A C T A G C A 4
- G A G G A C T G G G A T T A C T A G G G 4
+ A A G T G T G G G G A T T A C - A G G A 3
+ T G T T A A G G G G A T T A C T A G G A 2
+ T A T T A T G G G T A A T A C A A G G A in silico
- G A T T G G A G G G A T T A C T A G G A in silico
+ G A T T A T G G G G A T T A C A A T T A in silico
- G A T T A T T G G A A T T A C T A G G T in silico
- A A T T A T C T G G A T T A C A A G G G in silico
+ A A T T A T G A A G A A T T C T A G G A in silico
+ G A T T A T G G G G A T T A C C A G T T in silico
+ A G T T C T G G G A A T T A C T A G A A in silico
- A G A T A T G G G G G T T A C T A A G A in silico
- T A G T A T G G G A A T T T C T A G G A in silico
Sequence Alignments
More potential off-target sites identified through GUIDEseq
using highly modified gRNAs
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• gRNAs delivered at final concentration of 10 µM into HEK-293 cells expressing Cas9
Guide #1 Guide #2 Guide #3
% = sum of the total unique CRISPR-Cas9 reads
38% 12% 13%
2% 1% 4%
On- & off-target comparison of the gRNA forms
• gRNA variants targeting AR & EMX1
– Transfected by electroporation into HEK-293 cells stably expressing Cas9
• RNP (gRNA variants complexed to Cas9 nuclease [Alt-R WT Cas9 or
Alt-R HiFi Cas9])
– Transfected by electroporation into HEK-293 cells
– Alt-R electroporation enhancer included in transfection
• gDNA extracted after 48 hrs
• On- and off-target editing detected using rhAmpSeq system
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Experimental conditions:
On- & off-target assessment via rhAmpSeq system (AR target)
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• Similar off-target sites and OTE
levels detected across different
gRNA forms.
• Slightly higher OTE levels with
more modified gRNAs (remain
stable in the cell longer).
• OTE levels greatly reduced
when Cas9 delivered as RNP,
rather than expressed form.
• OTEs completely removed when
using Alt-R HiFi Nuclease, while
maintaining on-target editing.
On- & off-target assessment via rhAmpSeq system (EMX1 target)
30
• Similar off-target sites and OTE
levels detected across different
gRNA forms.
• Slightly higher OTE levels with
more modified gRNAs (remain
stable in the cell longer).
• OTE levels reduced when Cas9
delivered as RNP, but still present.
• OTEs greatly reduced when using
Alt-R HiFi Nuclease, while
maintaining on-target editing.
All gRNA and Cas9 forms tested produce identical Indel profiles
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gRNAs targeting EMX1
Conclusions
• Different gRNA formats give similar on-target editing when delivered as RNP.
(Higher modification needed when co-delivered with Cas9 in an expressed form)
• Subset of sequences respond better to higher modification, even with RNP delivery.
(Sequence and cell type dependent effect)
• Chemically synthesized gRNAs allow addition of chemical modifications, which
increase stability and reduce induction of innate immune system. (IVT sgRNAs induce
high levels of IFNα)
• The different gRNA forms result in the similar off-target editing levels at similar
sites. (Slight increase with gRNAs with increased modification)
• Delivering the gRNAs as RNP complexes shows a huge reduction in non-specific
editing. (This editing is even further reduced when using the Alt-R HiFi Cas9, while
maintaining high on-target activity)
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Alt-R CRISPR System — a complete workflow
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Design Cut Repair Analyze
Alt-R CRISPR-Cas9
guide RNA design tool
• Predesigned guides
• Custom designs
• Design checking
• Human, mouse, rat,
zebrafish, and
C. elegans
Coming soon –
HDR design tool
Alt-R guide RNAs
• Cas9 crRNA:tracrRNA
• Cas9 sgRNA
• Cas12a crRNA
Alt-R CRISPR proteins
• WT Cas9
• HiFi Cas9
• D10A nickase
• H840A nickase
• Cas12a (Cpf1)
Alt-R Electroporation
Enhancers
Ultramer
Oligonucleotides
• Up to 200 bases
Megamer ssDNA
Fragments
• Up to 2000 bases
• Sequence-verified
via NGS
Alt-R HDR Enhancer
Genome Editing
Detection Kit
• Simple, fast, T7EI-
based assay
Coming soon –
rhAmpSeq system for
CRISPR
• Multiplexed,
amplification for
Illumina NGS
• Up to 1000-plex
NEW: Alt-R HDR Enhancer increases HDR rates
with Cas9 and Cas12a nucleases
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0
10
20
30
40
50
60
70
MET TNPO3 site1 TNPO3 site 2 HPRT control HPRT site 1 HPRT site 2 HPRT site 3 HPRT site 4
Alt-R S.p. Cas9 nuclease v3 Alt-R A.s. Cas12a (Cpf1) nuclease v3
Eco
RI c
leav
age
(%)
Alt-R HDR Enhancer Neon electroporation, Jurkat cells
4 µM RNP, 3 µM HDR template
No treatment 30 µM HDR Enhancer
HDR enhancer is a small molecule compound that increases HDR efficiency
Take home messages• Standard Alt-R 2-part crRNA + tracrRNA works well for many
applications and is the most cost-effective synthetic option– Cas9 expressing cells
– gRNA screens (libraries)
– RNP delivery
– Reports of greater efficiency than sgRNA in zebrafish
• Increased chemical modifications in Alt-R crRNA XT + tracrRNA or using Alt-R sgRNA can add additional nuclease stability– Co-delivering with Cas9 plasmid or Cas9 mRNA
– Lipid nanoparticle delivery
– High nuclease environments
– Subset of sequence more susceptible to nuclease degradation
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More information and protocols available at: www.idtdna.com/crispr-cas9
(Click on the “Resources” tab for protocols)
THANK YOU
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reserved. Alt-R, Megamer, Ultramer, and rhAmpSeq are
trademarks of Integrated DNA Technologies, Inc., For
specific trademark and licensing information,
see www.idtdna.com/trademarks
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