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SureGuide
June 12, 2015
2
Fully Customizable Nuclease Specificity • Harness the power of the next-generation of genome editing tools
• Target any region of interest, including long, complex stretches of DNA
Synthesize gRNA on demand • High quality, high yield IVT kit assures reliable results
• Fast, simple, and easy to obtain new guides for different applications
Simple, validated reagents you can be Sure of • Faster start-up times with validated and purified reagents
• Optimized systems allows you to focus on the science, not the tools
Fast, Flexible, and Easy:
Purified Cas9 + gRNA synthesis system
All-in-One
crRNA
Spacer
(guide sequence)
crRNA processing
CAS9 CAS2 CAS1 csn1 CRISPR
dispensable
tracrRNA
The CAS9/CRISPR system targets double stranded DNA for cleavage
tracrRNA
..NNNNNNNNAGTAATATCAAAAAAGCCCCCTTGATTATCNGGNGNNN……
Repeat sequence Repeat sequence spacer
Genomic DNA
Cleavage site
TACGAGGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACAGTAATATCAAAAAAGCCCCCTTGATTATCGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAAC
PAM site
tracrRNA and crRNA can
be linked to mimic
processed form
For a 50% GC genome PAM
sites are expected to occur
1/32 base pairs
RNAse III
in vitro DNA cleavage by CAS9
stem (30 nt) spacer (20 nt)
tracr tail (13 nt)
CAS9
Needed for in-vitro cleavage
guide RNA (sgRNA)
CAS9
5’
5’ 3’
3’
OH
OH
?
?
5’
5’ 3’
3’ OH
OH
?
?
PAM
Mechanism of action
Agilent confidential
CAS9 binding of guide RNA
evaluation of PAM sites
Guide fitting
conversion to cleavage competent form
• spCAS9 does not turn over!
• It is not an enzyme in the
conventional sense but acts
as a site-specific cleavage
reagent
What are the current primary uses of CAS9?
CAS9
CAS9
Homologous recombination
targeted, concise insertion or replacement
CAS9 expression vector
Guide RNA
expression vector
CAS9
Purified CAS9 protein
purified guide RNA
Better with NLS on CAS9
NHEJ
• Leads to scar at cleavage site (indels)
• mutates target site
DNA oligo libraries
Libraries of guides
Libraries of inserts
Recommended reaction conditions
Reaction volume 20 µl
Target DNA 100 ng
Guide RNA 1 pmole
CAS9 enzyme 125 ng (798 fmoles)
Incubate for 1h at 30 °C,
heat inactivate for 15 minutes at 65 °C
Unit definition:
The amount of CAS9 required to cleave
50% of 100 ng linearized pKS-kanC1 using
the kanC1 guide under the above conditions
1U of CAS9 25 ng
Concentrations in unit definition assay:
guide RNA 50 nM
CAS9 protein 39.9 nM
Target DNA
• Target sites 2.6 nM • PAM sites 1.05 µM
CAS9 titration
guide titration
time course
Agilent confidential
Does spCAS9 display the expected PAM requirements?
Expected PAM site: …nnnnNGGNN…
Heat map of cleavage efficiencies: only
mutations of positions 2 and 3 affect cleavage
efficiency
Agilent confidential
Are cleavage products compatible with down stream molecular biology applications?
Expected product sizes:
3.9 kB, 180 bps
Cloning of 156 bps fragment
10 randomly picked clones analyzed by
restriction analysis
All clones were the expected product
Cloning of CAS9-digested DNA fragments
Cloned CAS9-excised 5.4 kb fragment
into Strataclone blunt vector
Selected for marker (gentamycin
resistance) encoded by cloned fragment
Picked 10 colonies for restriction
analysis
Analyzed cloning junctions of 5 isolates
Additional nucleotide at junction is
due to cleavage at the -4 position
instead of the -3 position relative to
the PAM site
Wobble cleavage occurs at a
frequency of 1/20
adaptors can be ligated to CAS9 digested sites
CAS9
P P
cleave target with CAS9
ligate adaptors with T4 DNA ligase
purify ligation product
PCR amplify ligation product
no
CA
S9
CA
S9
dig
este
d
SureGuide Ordering
Genome Editing for Functional Genomics
Studies the relationship between the genome
and its regulatory elements and the phenotype
(function & interaction)
Functional Genomics
CRISPR enables the easy and flexible implementation of
targeted functional assays for the entire Genome:
• KnockOut Exome
• KnockOut Inter-genes (Regulation)
• GE Activation (Regulation)
• GE Inhibition (Tunable RNA KnockDown, Regulation)
• KnockIns & Genome Tagging
CRISPR & Functional Genomics
gRNA libraries: Functional Genomics
gRNA Oligo Array Synthesis gRNAs cleaved &
PCR amplified
Cloning & Purification
Viral
packaging
Transduction
and selection of
Target Cells
Treatment
vs. Control
Screening and
Hit sequencing
Pathway analysis
(e.g. drug resistance
KO assay)
Early access to Agilent gRNA libraries
Catalog Libraries Format: Cloned in a lentiviral vector
• Exome Human
• Exome Mouse
Cloned Catalog Exome Libraries Paired Gene Libraries
Custom Custom Paired sub-libraries
(non-cloned sub-libraries)
Custom Probe sets –
Ready to clone (Human/Mouse)
• Custom Exome Pools
• Custom Mouse and Human
Genome Target sets
• Cloning kit
Custom gRNA sequences (Any Species)
Custom
(non-cloned)
Targeted Gene Editing
TC-RNA: Synthetic Guides in
single well, with modified
nucleotides
Cas9 and Guides in vivo
delivery methods
Agilent Confidential
-In development
-Early access coming soon
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