Download CRISPR/Cas9 - University of Leeds

6th April 2016
PAL workshop: Advances in Gene Editing Technology
University of Leeds
CRISPR/Cas9
Genome Editing Tools from NEB
Max Fritsch, PhD
Technical Account Manager
New England Biolabs UK
CRISPR/Cas9
1. Components of CRISPR/Cas9 system
2. CRISPR/Cas9 strategies
– Expression vectors
– sgRNA and Cas9 mRNA
– Cas9 protein
3. Screening for CRISPR/Cas9 mutations
Components of CRISPR/Cas9 Genome Editing
Cas9 nuclease
Target sequence with PAM
(Protospacer Adjacent Motif)
sgRNA:
~20 nt target-specific region
~80 nt invariable scaffold sequence
Main Applications for CRISPR/Cas9
•
Cas9 cleaves target sequence and
causes double strand break:
•
Cell repairs double strand break
•
Knock-outs:
introducing indels  disrupt genes
•
Knock-ins:
introducing targeted mutations with a
homologous donor DNA repair template
Genome Editing Strategies
Cultured Cells
Genome Editing Strategies
Embryo/ Zygote
sgRNA: What is it?
• crRNA targeting sequence:
– 20 nt sequence that is homologous to gene of interest
• tracrRNA sequence
– Constant ~80 nt sequence recruiting Cas9 nuclease
Genome Editing Strategies
Cultured Cells
Guide RNA: Working with Plasmids
• Cloning options to introduce new guide sequences:
•
Type IIS restriction enzymes:
– BbsI; BsmBI
– ssDNA oligosc
•
Site-Directed Mutagenesis
– End-to-end primers with
20 nt insertion
•
NEBuilder Hifi Assembly
– ssDNA oligo with
overlapping ends
Type IIS restriction enzymes
•
Type IIS RE cut outside recognition sequence  no RE site left
– ssDNA oligos
– Annealed with overhangs
– BbsI; BsmBI
– Ligation into vector
Genome engineering using the CRISPR-Cas9 system.
Ran, FA.*, Hsu, PD.*, Wright, J., Agarwala, V., Scott, DA., Zhang, F. Nature
Protocols Nov;8(11):2281-308. (2013).
Q5 Site-directed Mutagenesis kit
•
Quick; about 2h
•
Plasmids up to 20 kb
•
Master mixes
NEBuilder Hifi DNA assembly
•
ssDNA oligo
– 19-21 nt target sequence
– 25 nt vector overlaps
•
Doesn’t require RE sites!
Genome Editing Strategies
Embryo/ Zygote
Guide RNA: in vitro Synthesis
•
HiScribe™ T7 Quick High Yield RNA Synthesis Kit
– NTP buffer master mix
– T7 RNA Pol master mix
– Control template
– Dnase I
– LiCl solution
Guide RNA: in vitro Synthesis
• Generating Templates for RNA in vitro Transcription:
– ssOligos: annealing and fill in with T4 DNA polymerase or Klenow
fragment
Guide RNA: in vitro Synthesis
Genome Editing Strategies
Embryo/ Zygote
Cas9 mRNA synthesis
• Structure of mRNA
– 5’-m7G-cap
– 3’-poly(A) tail
• Cap and poly(A) tail are crucial for
mRNA stability and expression
Cas9 mRNA synthesis
•
HiScribe™ T7 ARCA mRNA Kit (E2065)
– For poly(A)-tail in DNA template
•
HiScribe™ T7 ARCA mRNA Kit (with tailing)
(E2060)
– Enzymatic tailing with Poly(A) polymerase
Cas9 Protein – Why?
Cell culture
Zygote/Embryo
in vitro
Kim et al 2014:
• “RGEN ribonucleoproteins (RNPs) induce site-specific
mutations at frequencies of up to 79%, while reducing offtarget mutations associated with plasmid transfection…”
• “RNP delivery is less stressful to human embryonic stem cells,
producing at least twofold more colonies than does plasmid
transfection”
Kim et al 2014:
Figure 2.
“... RNP delivery was less toxic to ES cells,
producing at least twofold more colonies
than did plasmid transfection ...”
Figure 6.
“... RNPs cleaved chromosomal DNA almost immediately after delivery ...”
“... Cas9 protein was rapidly degraded in cells when delivered directly ...”
“... Cas9 protein was expressed from plasmid for several days ...”
Cas9 Protein
• Cas9 Nuclease NLS, S. pyogenes
– C-term. NLS (SV40 T antigen)
– recommended for in vivo use
• Cas9 Nuclease, S. pyogenes
– without NLS
• High concentration: 20 μM (48 μg, 96 μg)
• Low concentration : 1 μM (11 μg, 56 μg)
Cas9 Protein – in vitro
• Testing Efficiency of sgRNA in vitro
• Screening for mutations!
Online protocol on the NEB website!
Screening for mutations
•
How to screen for mutated clones?
•
Homology Directed Repair:
– Usually inserting new seuquence
– PCR and gel electrophoresis
•
Non-homologous End Joining:
– Random insertion or deletion of a few
bp  too small for PCR and gel
– RE and T7 Endonuclease I
Screening with T7 Endonuclease I
Evaluating Targeting Efficiency with T7 Endonuclease I
Protocol:
• Amplify modified locus
• Denature WT and mutant alleles
• Digest mismatched Mut/WT
duplexes
• Analyse on fragment analyser or
agarose gel
 Digestion of edited loci only
 Detects 5% to 95% mutant loci
Screening with T7 Endonuclease I
“…we find that T7E1
outperforms the Surveyor
nuclease in terms of sensitivity
with deletion substrates,
whereas Surveyor is better for
detecting single nucleotide
changes. We conclude that
T7E1 is the preferred enzyme
to scan mutations triggered by
engineered nucleases.”
EnGen™ Mutation Detection Kit
Complete kit for Amplification, Detection and Analysis:
E3321 Components (25 reactions):
• EnGen™ T7 Endonuclease I
• Q5 Hot Start High-Fidelity 2X Master Mix
• Control Template and Primer Mix
Screening with Restriction Enzymes
•
•
•
•
Alternative or complimentary assay to T7 Endonuclease I
Digestion of unedited sequences
Single base sensitivity
Easy workflow – most NEB RE work in a PCR buffer
PAM
5’...NNNNNNNNNNNNNNNNN|NNNNGG...3’
3’...NNNNNNNNNNNNNNNNN|NNNNCC...5’
BslI
Check sequence online
with NEBcutter
MwoI
5’...CCNNNNN|NNGG...3’
3’...GGNN|NNNNNCC...5’
5’...GCNNNNN|NNGC...3’
3’...GGNN|NNNNNCC...5’
EcoNI
5’...CCTNN|NNNAGG...3’
3’...GGANNN|NNTCC...5’
BstXI
5’...CCANNNNN|NTGG...3’
3’...GGAN|NNNNNTCC...5’
Analysis by Sequencing
• Characterise mutations by sequencing:
– For gene disruption – what sequence has been inserted/deleted?
Is it a frame shift?
– For Knock-in – is the sequence inserted/changed correctly
• Sanger Sequencing:
– Monarch PCR & DNA Cleanup Kit
– Rapid PCR Cleanup Enzyme Set
• Next Generation Sequencing:
– Characterise off-target effects
– 100% certainty
– NEBNext reagents for Illumina and IonTorrent