iGEM Projects PubMed Entries per 100 000 2 Cas as9 Protein - PowerPoint PPT Presentation

Exponential increase of scientific interest in CRISPR-Cas9 iGEM Projects PubMed Entries per 100 000 2

Cas as9 Protein cleaves DNA Target DNA PAM Sit ite allows sg sgRNA binds target DNA Cas9 binding 3

“I was standing there with some biologists... and they started to lose their s---, like genuinely lose their s--- , about this thing called CRISPR.” Jad Abumrad, Radiolab, Antibodies Part 1: CRISPR 4

sgRNA Efficiency PAM Seq equence Different sgRNA have Only sgRNA target with difficult-to-predict an adjacent NGG PAM biochemical efficiencies site can be chosen 5

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Re-engineering sgRNA structure with restriction sites that allow target sequence exchange 7

Re-engineering sgRNA structure with restriction sites that allow target sequence exchange Original sgRNA Modified sgRNA Re-engineering sgRNA structure with restriction sites that allow target sequence exchange 8

sgRNA Targeting Nucleotide Preference Reproduced from Rational design of highly active sgRNAs for CRISPR-Cas9 – mediated gene inactivation, Figure 3. Doench et al. , 2014 sgRNA optimization still requires testing in the lab e.g. Wang et al. 2015 9

Original sgRNA Modified sgRNA sgRNA secondary structure modules determined by Briner et al. 2014 10

Original sgRNA: Expect low RFP targeting RFP +sgRNA Modified sgRNA: targeting RFP Expect low RFP +sgRNA Control: Used BBa-K1645999 without dCas9 Expect high RFP 11

Identical decrease in RFP expression with each sgRNA structure Original sgRNA Modified sgRNA 12

• Designed a new sgRNA scaffold that allows easy exchange of target sites • Verified that the scaffold does not interfere with dCas9 binding and expression control • Foundation of a functional prototype, next steps include full verification of exchange 13

Overcoming NGG PAM limit on target selection through molecular dynamics models 14

Modeling background: 3D protein structure Found a 3D dCas9 structure (Nishimasu et al . 2014) Modeled using PyRosetta molecular dynamics software 15

Suite of Python scripts to rank PAM specificity for Cas9 mutants Generate 256 Determine Cluster and Mutate Cas9 PAM variants binding score rank scores structure 27.1 AAAA 27.3 AAAC    -10.1 AAAG 27.8 AAAT ... ... 200.6 TTTT 16

Docking energy minimization as a measure of binding affinity 17

Available data: Cas9 with altered PAM specificity N Ju June 2015: G Empirical validation: Cas9 G mutants that bind NGAG N and NGA PAMs found by Kleinstiver et al. ( Nature , N 2015) using directed G evolution A G 18

Wild-type reproduces fairly well, mutant less so Wild-type NGAG Mutant R = 0.4 .46 R = 0.6 .66 p = 1.1 .1e-14 14 p = 2.2 .2e-16 16 NGGN NGNG 19

Investigating binding profile of Kleinstiver mutant dCas9 + + NGG NGAG TARGET TARGET 20

Mutant dCas9 does not produce expected binding profile 50000 45000 40000 35000 GFP Intensity GFP Intensity 30000 25000 20000 15000 10000 5000 0 NGG NGAG NGG PAM Site NGAG PAM Site 21

• Explored dCas9-DNA binding affinities in silico • Created framework for testing sgRNAs computationally • Results suggest more work and a reassessment of assumptions • Framework & documentation available online 22

sgRNA Efficiency PAM Seq equence Applications: gene editing gene drives expression control 23

CRISPieR Application: engineered plants with CRISPR-Cas9 antiviral defense 24

Model Virus: Model Plant: Cauliflower Mosaic Virus (CaMV) Arabidopsis thaliana 25

How does CaMV affect plant cells? CaMV Production without CRISPR Defense 26

How does CaMV infection spread among plant cells? • NetLogo models available on • Agent-based stochastic modeling • Incorporates intracellular ODEs via Euler’s method 27

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Using protoplasts as a model  29

Can we express our CRISPR-Cas9 defense system in plant cells? Trial #2 Trial #1 30

Mathematical models are not bound by the same constraints  31

How does CRISPR-Cas9 deactivate viral genomes? 32

Results for Single Cell Result s for Plant Tissue 33

Could improved sgRNA efficiency or PAM flexibility aid defense? CRISPieR (t 1/2 = 0.9h) CRISPR (t 1/2 = 1.8h) 34

CRISPR Plant Defense 35

CRISPR Plant Defense → CRISPieR Plant Defense 36

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Do people respond differently to different wording? Ran a survey at the university and a local farmer’s market 121 121 Person sample size Similar questions using the words: GMO versus 60 60 GMO surveys CRISPR-Cas9 Technology 61 61 Gene editing surveys 38

Do people respond differently to different wording? How do you feel about eating food with [GMO or CRISPR-Cas9] ingredients? 39

Why do people oppose the sale of GM foods? 40

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W OULD YOU SUPPORT SCIENTIFIC RESEARCH INTO THE FIELD OF GM FOODS ? YES 96% 42

CRISPieR Cas9 PAM Flexibility Simple sgRNA Exchange • Python suite using PyRosetta • Added restriction sites to sgRNA • Correlated affinities for wild type scaffold • Inconclusive results for NGAG and • Showed modified scaffold didn’t NGAN PAMs affect dCas9 activity • Potentially due to different dCas9 CRISPieR Application Gold Requirements • Additional Practices work (IP, business) CRISPR Plant Defense • Cas9 expression in Arabidopsis • uOttawa, Aalto-Helsinki collaborations protoplasts • Improved characterization of xylose • Viral models suggesting system will (BBa_K1323014 and BBa_K1323002) work • Models also suggest CRISPieR will work 43

Elementary School Commercialization iGEM Academy iGEM Critique Digest Calculator High School PyRosetta Suite Floral Dip University + GitHub 44

Dr. Marc Aucoin Radmila Kovac Cherry Chen Julia Manalil Maye Saechao Dr. Trevor Charles Dr. Jiujun Cheng Dr. Aiming Wang Dr. Andrew Doxey Maya D’Alessio Destin Sigurdson Dr. Barbara Moffatt John Heil Jamie McNeil Dr. Brian Ingalls Kathy Lam Lauren Kennedy Dr. Simon Chuong Suzie Alexander Dr. Susan Lolle Pavel Shmatnik Dr. Pearl Chang Dragos Chiriac 45

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1. Briner, A. E., et al ., Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality, Molecular Cell , vol. 56, no. 2, pp. 333-339 (Oct. 2014). 2. Kleinstiver, B. P., et al . Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature , 523, pp. 481-485 (Jun. 2015). 3. Nishimasu, H. Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA. Cell , 156, pp. 935-949, (2014). 4. Doench, J. G., et al . Rational design of highly active sgRNAs for CRISPR- Cas9 – mediated gene inactivation. Nature Biotechnology , 32, 12, pp. 1262 – 1267 (Sep. 2014). 5. Wang, X. et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci. Rep. 5, 13348; doi: 10.1038/srep13348 (2015). 47