Rewriting the Genome With gblocks Gene Fragments

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1 Rewriting the Genome With gblocks Gene Fragments Harnessing the Power of CRISPR and Synthetic Biology Adam Clore, PhD Manager, Synthetic Biology Design and Support

2 Defining Synthetic Biology The design, redesign, and construc1on of new biological parts, devices, and systems

3 Genome Modification Using the Repair of Double Stranded Breaks In most eukaryotes non homologous end joining (NHEJ) is the most efficient DBS repair pathway Error prone, often creates INDELs The presence of homologous template (aka Donor DNA ) can induce recombination Efficiency and length of homology arms varies from one cell line to another

4 Recent Evolution of Genome Editing Zinc Finger Nucleases (ZNFs) TALENs Meganucleases CRISPR/Cas9 Curtin (2012), Plant Gen., 5, p42-50

5 Overview of Three Generations of Programmable Nucleases Cost Reliability Accuracy Zinc Finger Nucleases $$$$ Low Poor TALENs $$$$ High Good CRISPR $ High Good

6 CRISPR Easy Genome Modification Clustered Regularly Interspaced Short Palindromic Repeat A prokaryo1c defense mechanism that screens for and cleaves specific DNA sequences Can be used to create targeted changes to the genomes of bacteria, archaea, and eukaryotes

7 The 3 Stages of CRISPR Resistance Stage 1: CRISPR Adaptation Foreign DNA is incorporated in the CRISPR array. Stage 2: CRISPR Expression CRISPR RNAs (crrnas) are transcribed from the CRISPR locus. Stage 3: CRISPR Interference Foreign nucleic acid complementary to the crrna is neutralized.

8 CRISPR Applications New England Biolabs

9 How Do You CRISPR? CRISPR System Dual-expression plasmids Cas9 under constitutive promoter grna under Pol III promoter (U6/H1) Two single-expression plasmids Lentiviral transfection Microinjection Cell lines expressing Cas9 Alternative delivery of CRISPR components grna expression cassette (i.e., gblocks Gene Fragments) grnas Cas9 protein Alternative PAMs Features Most common, most published data Large plasmids (8-10 kb), tricky transfection Cloning of grnas is cumbersome Smaller plasmids Easier cloning of grnas Lower transfection rates High efficiency Requires cloning and pseudovirus production Preferred for embryos Highest efficiency (>95% of cells express Cas9) Requires specialized equipment and methods Eliminates variation in large plasmid transfection In search of increased targeting efficiency and reduced off-target effects

10 CRISPR Mediated Gene Disruption CHO cells In zebrafish In yeast The list goes on

11 CRISPRi CRISPR Based Gene Silencing

12 Method CRISPRi uses a nuclease dead Cas9 Protein to sterically block transcription elongation or promoter binding First demonstrated in E. coli by Qi et al (Weissman Lab, Berkeley) in

13 Transcription Repression

14 Benefits of CRISPRi Functions in all domains of life sirna is not active in prokaryotes and some fungi No permanent change to genome Different activity than RNAi Anecdotal information suggests that CRISPR may be more robust that RNAi

15 Screening With CRISPR Libraries First large screen done by Zhang Lab in 2014 Created genome-scale CRISPR knockout library (GeCKO) Lentiviral vector with CRISPR cassette Identification of genes associated with vemurafenib sensitivity (B-Raf inhibitor) 18,080 genes 64,751 grnas Science (2014), 343

16 Screening With CRISPR Libraries First large screen done by Zhang Lab in 2014 Created genome-scale CRISPR knockout library (GeCKO) Lentiviral vector with CRISPR cassette Identification of genes associated with vemurafenib sensitivity (B-Raf inhibitor) 18,080 genes 64,751 grnas

17 CRISPR as a Biological Sensor Paris-Bettencourt igem team 1 st place in 2013 overgrad competition Detection of tuberculosis drug resistance genes using a phage-delivered cassette containing: a Cas9/gRNA targeting a drug resistance gene a LacZ gene driven by an SOS dependent promoter

18 CRISPR as a Biological Sensor Designed for a quick and inexpensive field diagnostic

19 Creating Long Accurate Synthetic DNA Without Cloning IDT introduced the concept of synthetic gene fragments bp in length Sequence-verified Short delivery time and low price 200 ng provided, dry Top 3 questions: - Can you make them longer? - Can you make them variable? - Can I get a discount? High quality DNA fragments, Fast assembly and cloning required

20 Using gblocks Gene Fragments for CRISPR Current Protocols in Molecular Biology (2014),

21 Using gblocks Gene Fragments for CRISPR Current Protocols in Molecular Biology (2014),

22 gblocks Gene Fragments for CRISPR

23 Design of Donor DNA dsdna typically requires homology arms >500 bp in mammalian cells Caution! HR efficiency and optimal arm length varies greatly between cell lines and must be experimentally verified ssdna can efficiently recombine with base homology arms TARGET GENE 5 Arm URA3 3 Arm

24 gblocks Gene Fragments 2015 We made them longer up to 2 kb gblocks Gene Fragments Usually Shipped (BD) Pricing We made them variable >50 citations bp 2 4 $89.00 USD bp 2 4 $ USD bp 3 5 $ USD bp 5 8 $ USD bp 5 8 $ USD bp 5 8 $ USD bp 5 8 $ USD

25 Surveyor Detection of CRISPR Modifications sgrna + Cas9 (A) CRISPR sgrna delivered to cells. (B) sgrna + CAS9 bind and cut their target sequence creating a double-strand break (DSB) in a portion of the cells. (C) Aberrant repair of some DSBs by non-homologous end joining (NHEJ) results in insertion, deletion or substitution (depicted by red X).

26 Surveyor Detection of CRISPR Modifications (C, D) Genomic DNA is harvested from the transfected pool of cells and amplified at the locus of interest. (E, F) PCR product is denatured and re-annealed creating heteroduplex formation between wild type and modified amplicons. 26

27 Quantitative Assessment of CRISPR Gene Editing via Mismatch Endonuclease Average % Cleavage of Biological Triplicates via Fragment Analyzer

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29 Biosecurity IDT is one of the five founding members of the International Gene Synthesis Consortium (IGSC) Screens the sequence of every gene and gblocks Gene Fragment order To ensure safety and regulatory conformance IDT reserves the right to refuse any order that does not pass this analysis For more information about the IGSC and the Harmonized Screening Protocol, please visit the website at In October of 2010, the United States government issued final Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic genes should perform gene sequence and customer screening. IDT and the other IGSC member companies supported the adoption of the Screening Framework Guidance, and IDT follows that Guidance in its application of the Harmonized Screening Protocol. For more information, please see 75 FR (Oct. 13, 2010), or

30 Additional Resources CRISPR Resources Information for gblocks Gene Fragments Support for Help With Design, Experimental Issues, and Ordering Other Educational Resources at Under Support & Education Menu: DECODED Newsletter ( DECODED) Video Library Frequently Asked Questions More Integrated DNA Technologies: Coralville, IA San Diego, CA Leuven, Belgium Singapore 30 INTEGRATED DNA TECHNOLOGIES

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