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Mike BoxemMihail Sarov
CRISPR/Cas9-based genome engineering…. an update
Workshop overview
• Design sgRNA and repair template
• Inject P0– Cas9– sgRNA– Repair template
• Select mutants or transgenics– Phenotype– Fluorescence– PCR
Bacterial immunity
Large space for improvement and additional applications
A single guide RNA can direct dsDNA cleavage by Cas9
Jinek et al. Science. 2012 Aug 17;337(6096):816-21
Gasiunas et al. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39)
Experimental procedure
• Design sgRNA and repair template
• Inject P0– Cas9– sgRNA– Repair template
• Select mutants or transgenics– Phenotype– Fluorescence– PCR
Target site selection
• Only one requirement: PAM sequence (NGG) has to bepresent
• When using U6 promoter expression avoid UUUU stretches as they serve as Pol III terminators
• U6 transcription initiation requires a 5’G – can add an extra G to the guide G(N)20NGG
• In vitro transcription requires polymerase specific initiating nucleotides– For T7 GG is optimal <can be added to the guide GG(N)
ON-target efficiency
• Multiple methods to predict efficiency (of limited value) Doench et al. 2014; Wang et al. 2014; Farboud and Meyer 2015; Xu et al. 2015
• (N)18GGNGG works better than (N)20NGG <but is rare - use is practical for deletion experiments - very rarely useful for HDR (Farboud and Meyer 2015)
• ‣(N)19CNGG is disfavoured in a consensus derived from a large dataset (Doench et al.)
Alternative PAM sequences
Fire lab
Modified Cas9
Cpf1
An improved sgRNA design
Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G.-W., et al. (2013). Dynamic Imaging of Genomic Loci in LivingHuman Cells by an Optimized CRISPR/Cas System. Cell, 155(7)
OFF-target effects
• Appears to be a negligible problem in C. elegans• Additional GGs at the 5’ end reduce the off-target activity
(Cho, S.W. et al. 2014; Kim, D. et al. 2016)• Shorter guides (17) reduce off target activity at a minor on
target penalty (Fu, Sander, Reyon, Cascio, Joung, Nat. Biotech. 2014 Mar; 32(3):279-84)
• Recommended: Outcross and compare independent lines
Modified fidelity Cas variants
Cas9n
dCas9-FokI
Need 2 cuts to create DSB
Need to target 2 sites, and dimerize FokI
High fidelity variants that bind DNA less strong
! May not tolerate an extra 5’ G in the sgRNA
SpCas9-HF (Joung lab)eSpCas- (Zhang lab)
Experimental procedure
• Design sgRNA and repair template
• Inject P0– Cas9– sgRNA– Repair template
• Select mutants or transgenics– Phenotype– Fluorescence– PCR
Genome engineering strategies
Oligo-mediated repair SEC vectorsSAP/TRAP vectors
Random mutationsDeletions (use 2 sgRNAs)
Oligonucleotice mediated repair(Alexandre Paix/Seydoux lab)
• Use for– Point mutations and small insertions (up to ~ 140 bp based on longest
available oligo)– Precise gene deletions (use 2 sgRNAs)
• Design considerations– ~35 bp arms (longer arms are detrimental)– Try to have cut site close to intended mutations– Introduce mutations to prevent re-cutting (mutate PAM if possible, if
not use silent mutations)– Add additional changes for detection: use codon table– PAGE purification increases efficiency
Alaggc
agaagtatggatcctattcgcgaaggagaagtggcccacgagggaagctctttcacgaagctcgaatacggcggcgaaggaacatacgggtttacacactcaacgacgaaggagSer
Add mutations between cut-site and edit point!
atcctattcgcgaaggagaagtggcccacgagggaGGctctttcacCaaATtAgaGtaTggcggcgaaggaacatacgggtttacacactcaacS F T K L E Y
Genome sequence target site PAM
ssODN repair template35 bp35 bp
Desired change Conservative changes
Recombination consistently occured here
Strand preference
• The DNA strand not targeted by the sgRNA is more accessible
• ssODNs that anneal to the non-target DNA strand are more efficient
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., & Corn, J. E. (2016). Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nature Biotechnology.
Genome engineering strategies
Oligo-mediated repair SEC vectorsSapTrap vectors
SEC vectors by Dickinson et al.
Gibson cloning of SEC vector repair template
SapTrap vectors by Matthew Schwarz and Eric Jorgensen
Modular assembly using SapI enzyme
5’-GCTCTTCNNNN---3’-CGAGAAGNNNN---
SEC/SapTrap cloning considerations
• First amplify a larger region containing both homology arms– Good template for PCR with Gibson tails on primers– Provides outside primers for identification of engineered
animals
• Introduce mutations that disrupt the recognition sequence!
• Midipreps of SEC vectors are hard! We use Macherey-Nagel low copy protocol with 200 ml culture, grown at 30°C.
Delivery of Cas9 and sgRNA
Plasmid-based (may be most suitable for long-arm HDR)
RNA
Ribonucleoprotein (may be most suitable for short-arm HDR)
Experimental procedure
• Design sgRNA and repair template
• Inject P0– Cas9– sgRNA– Repair template
• Select mutants or transgenics– Phenotype– Fluorescence– PCR
Identifying engineered animals
• Phenotype• PCR• Co-CRISPR/co-conversion• Selectable markers
Phenotype-based selection
• Most modification probably can’t be identified by phenotype, but….
• Mutations can cause visible phenotypes
• Fluorescent insertions can be identified on low power binoculars
PCR-based identification
Mismatch detecting nuclease (CelI, T7E1, surveyor)
Size difference
PCR-based identification
tcctattcgcgaattcgaagtggccc
tcctattcgcgaaGtcgaagtggccc
tctttcacgaagctcgaatacgg
tctttcacCaaATtAgaGtaTgg
Restriction fragment length polymorphism
Silent mutations
Selection markers
Co-CRISPR(Arribere et al. 2014, Kim et al. 2014, Ward 2015)
Site 1 (co-CRISPR) Site 2 (desired change)
dpy-10(cn64) gain of functionsqt-1(e1350) dominant Rollerpha-1(e2123) rescue
Screen second site by PCR
Co-CRISPR considerations
• Best for efficient desired modifications– ssODN repair– Active sgRNA– Close to cut site
• Screen worms with jackpot broods• pha-1 repair easy to select, but a non-N2
background
Positive selection markers
unc-119 rescueblasticidin resistancehygromycin resistanceneomicin resistance
Positive selection advantages
• Large homology arms are less sensitive to distancefrom cut site
• Recombination can take place in F1 germline as well as P0
• Even rare events are detected, as all animals are screened.
Positive selection marker considerations
• Counter selection against extrachromosomal array appears to be unneeded with hygR
Flowchart from Dickinson et al. Genetics 2016
Other uses of CRISPR/Cas9
Modifying gene expression
dCas9
dCas9
Interfere
Activate
Chromatin IP or visualization
Visualization
Purification
The next CRISPR?
Questions/discussion