Designing CRISPR mediated Gene disruptions with gblocks Gene Fragments Adam Clore, PhD Manager, Synthetic Biology Design Integrated DNA technology
Typical CRISPR timeline in Mammalian cell lines Design Targets Order Reagents Clone grnas Transfection and selection Verify Genotype Select lines and propagate 2-8 days 2-5 days 2-8 days 4-12 days 4-8 days
Typical CRISPR timeline in Mammalian cell lines Design Targets Order Reagents Clone grnas Transfection and selection Verify Genotype Select lines and propagate 2-8 days 2-5 days 2-8 days Reagents Needed 1. Plasmids 2. grna targets 3. Screening oligos 4. Enzymes 4-12 days 4-8 days
Cas9 Delivery Methods Single Plasmid Systems Most common, most published Data Large plasmids (8-10 kb), tricky transfection Cloning of grnas is cumbersome Dual plasmid Systems Smaller plasmids Easier cloning of grnas Lower transfection rates Lentiviral transfection High efficiency Requires cloning and psudeovirus production Microinjection Preferred for embryos Highest efficiency (>95% of cells express Cas9) Requires specialized equipment and methods
Where to get your Cas9 Plasmids? For Model Organisms Addgene For Custom Cas9 Genes IDT! Codon optimization available Most likely will require subcloning
Target Design Resources CRISPR design---zhang lab ECRISP---dkfz (German Cancer Research Center) ZiFiT---Zinc finger consortium Cas9 Design---PKU Bioinformatics Broad Institute CasOT CHOPCHOP (Harvard) 6
What Data Supports Target Design? Looked at single and multiple mismatches within different target sequences in the EMX1 gene using HEK 293 cells Deep sequencing of 117 potential off target sites within the genome 7
What Data Supports Target Design? Some additional information on of target erects A great resource for mammalian CRISPR protocols 8
Zhang lab CRISPR design Input options: 25-250 nt sequence Target genome Batch option available (file in FASTA format) Design parameters: 20 bp followed by NGG PAM Scans for possible off target matches throughout selected genome Output format: Ranks output guides on scale of 1-100, with 100 being best Ordered by faithfulness of on target activity 100% minus weighted sum of off target hit-scores in the target genome Off target hit scores are displayed for top matches of each guide http://crispr.mit.edu/ Takes into account total number of mismatches, mismatch absolute position (mismatches near PAM have relatively high disturbance), and mean pairwise distance between mismatches
http://crispr.mit.edu/
http://crispr.mit.edu/ http://crispr.mit.edu/guide s/3730938747354219
more information from website http://crispr.mit.edu/
gblocks as grnas Over 14,000 grnas produced Rapid TAT Recommended by the Church Lab
grna gblocks in HEK cells Cas9 Plasmid plus linear gblocks Gene Fragments Protocol available and tested by IDT in Cas9 expressing HEC 293 cells 38646 AS 38673 AS 38674 AS No grna U9-Trcr NC1 Reagent grna Only Cells Only
What are gblocks Gene Fragments? 2012 IDT introduced the concept of synthetic gene fragments 125-2000 bp in length Sequence-verified Short delivery time and low price 200 ng provided, dry High quality DNA fragments, Fast - Assembly and cloning required
gblocks Gene Fragments 2014 Pricing >100 citations >70 CRISPR citations Typical grna Length gblocks Gene Fragments Usually Shipped (BD) Pricing 125-500 bp 2 4 79,00 EUR 501-750 bp 2 4 109,00 EUR 751-1000 bp 3 5 129,00 EUR 1001-1250 bp 5 8 179,00 EUR 1251-1500 bp 5 8 215,00 EUR 1501-1750 bp 5 8 249,00 EUR 1751-2000 bp 5 8 285,00 EUR
QC- three independent metrics 1. Size verification 2. Mass Spectrometry Sequence identification 3. Sanger sequencing
Design of Donor DNA dsdna typically requires homology arms > 500 bp in mammialian cells Caution! HR efficiency and optimal arm length varies greatly in cell lines and must be experimentally verified ssdna can efficiently recombine with 40-50 base homology arms TARGET GENE 5 Arm URA3 3 Arm
Typical CRISPR timeline in Mammalian cell lines Design Targets Order Reagents Clone grnas Transfection and selection Verify Genotype Select lines and propagate 2-8 days 2-5 days 2-8 days 4-12 days 4-8 days
Cloning Methods Cationic Lipids We prefer Lipofectamine RNAiMax Electroporation Zhang lab uses Amaxa P3 primary cell 4D Nucleofector kit Transfection For Lentiviral CRISPR systems Microinjection Embryos and cells not compatible with other methods
Typical CRISPR timeline in Mammalian cell lines Design Targets Order Reagents Clone grnas Transfection and selection Verify Genotype Select lines and propagate 2-8 days 2-5 days 2-8 days 4-12 days 4-8 days
Screening CRISPR Surveyor Assay Standard for identifying INDELs Requires primers for Next Gen Sequencing Gold Standard Enrichment