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

Jul 09, 2023 366 likes •853 views

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

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

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

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“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

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

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

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

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

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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

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Original sgRNA Modified sgRNA

sgRNA secondary structure modules determined by Briner et al. 2014

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11 +sgRNA +sgRNA

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

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

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  • 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

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Overcoming NGG PAM limit on target selection through molecular dynamics models

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Found a 3D dCas9 structure (Nishimasu et al. 2014) Modeled using PyRosetta molecular dynamics software

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Modeling background: 3D protein structure

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Suite of Python scripts to rank PAM specificity for Cas9 mutants

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Mutate Cas9 structure Cluster and rank scores Generate 256 PAM variants AAAA AAAC AAAG AAAT ... TTTT Determine binding score 27.1 27.3

  • 10.1

27.8 ... 200.6

 

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Docking energy minimization as a measure of binding affinity

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Available data: Cas9 with altered PAM specificity

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Ju June 2015: Empirical validation: Cas9 mutants that bind NGAG and NGA PAMs found by Kleinstiver et al. (Nature, 2015) using directed evolution

N G G N N G A G

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Wild-type NGAG Mutant

NGGN

Wild-type reproduces fairly well, mutant less so

NGNG

R = 0.4 .46 p = 1.1 .1e-14 14 R = 0.6 .66 p = 2.2 .2e-16 16

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Investigating binding profile of Kleinstiver mutant dCas9

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NGG

+ TARGET

NGAG

+

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5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 NGG PAM Site NGAG PAM Site GFP Intensity

Mutant dCas9 does not produce expected binding profile

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NGAG NGG

GFP Intensity

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  • 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

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sgRNA Efficiency PAM Seq equence

Applications: gene editing gene drives expression control

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CRISPieR Application: engineered plants with CRISPR-Cas9 antiviral defense

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

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How does CaMV affect plant cells?

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CaMV Production without CRISPR Defense

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  • NetLogo models available on
  • Agent-based stochastic modeling
  • Incorporates intracellular ODEs via

Euler’s method

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How does CaMV infection spread among plant cells?

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Using protoplasts as a model

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Can we express our CRISPR-Cas9 defense system in plant cells?

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Trial #1 Trial #2

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Mathematical models are not bound by the same constraints

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How does CRISPR-Cas9 deactivate viral genomes?

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Results for Plant Tissue

Results for Single Cell

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Could improved sgRNA efficiency or PAM flexibility aid defense?

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CRISPR Plant Defense

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CRISPR Plant Defense → CRISPieR Plant Defense

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121 121 Person sample size 60 60 GMO surveys 61 61 Gene editing surveys

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Do people respond differently to different wording?

Similar questions using the words:

GMO versus CRISPR-Cas9 Technology

Ran a survey at the university and a local farmer’s market

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Do people respond differently to different wording?

How do you feel about eating food with [GMO or CRISPR-Cas9] ingredients?

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Why do people oppose the sale of GM foods?

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WOULD YOU SUPPORT SCIENTIFIC

RESEARCH INTO THE FIELD OF GM FOODS?

YES 96%

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CRISPieR Application

CRISPR Plant Defense

  • Cas9 expression in Arabidopsis

protoplasts

  • Viral models suggesting system will

work

  • Models also suggest CRISPieR will work

Gold Requirements

  • Additional Practices work (IP, business)
  • uOttawa, Aalto-Helsinki collaborations
  • Improved characterization of xylose

(BBa_K1323014 and BBa_K1323002)

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CRISPieR

Simple sgRNA Exchange

  • Added restriction sites to sgRNA

scaffold

  • Showed modified scaffold didn’t

affect dCas9 activity

Cas9 PAM Flexibility

  • Python suite using PyRosetta
  • Correlated affinities for wild type
  • Inconclusive results for NGAG and

NGAN PAMs

  • Potentially due to different dCas9
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Elementary School High School University iGEM Academy Digest Calculator PyRosetta Suite + GitHub Commercialization iGEM Critique Floral Dip

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  • Dr. Marc Aucoin
  • Dr. Trevor Charles
  • Dr. Andrew Doxey
  • Dr. Barbara Moffatt
  • Dr. Brian Ingalls

Radmila Kovac Julia Manalil

  • Dr. Jiujun Cheng

Maya D’Alessio John Heil Kathy Lam

  • Dr. Simon Chuong
  • Dr. Susan Lolle
  • Dr. Pearl Chang

Cherry Chen Maye Saechao

  • Dr. Aiming Wang

Destin Sigurdson Jamie McNeil Lauren Kennedy Suzie Alexander Pavel Shmatnik Dragos Chiriac

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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).

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