July 17 - 19, 2017

Monday - Wednesday

Germantown, MD

The Bioscience Education Center

9:00am-5:00pm

1 Hour Lunch Break

This rigorous three day program is ideal for basic research and translational biology scientists who are looking for a balanced theoretical vs. hands-on introduction to gene editing iPSCs using CRISPR/Cas9. In this workshop, participants will be able to design CRISPR/Cas9 gene editing tools, deliver gene-editing tools into human iPSCs, and detect gene-editing events by PCR/ Surveyor Assay.

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Lecture and Hands-on Interactive Training
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Team taught by active researchers
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Comprehensive binder containing workshop material
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Space limited to 15 participants
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Registration Fee: $895

Course Co-Director

Serguei Kozlov, PhD, MBA, PM
Team Leader, Principal Scientist and Program Manager, Center for Advanced Preclinical Research, Leidos Biomedical Research, Inc

Course Co-Director

James Kehler VMD, PhD
Director of Scientific Alliances, MTI-GlobalStem

Lectures:

  1. Basic Principles of Genome Editing Technologies from ZFN to CRISPR
  2. Special Aspects of CRISPR/Cas9 Technology
    • Structural Aspects of CRISPR/Cas9 Target Recognition and Biologic Patterns Among the Family of Bacterial Targeted Endonucleases
    • Dichotomy of Alternative Mechanisms for Double Stranded DNA Break Repair and How They Can Be Manipulated to Benefit the Desired Outcomes of a CRISPR/Cas9 Experiment
    • CRISPR vs. RNAi Technologies: Strengths, Weaknesses, Opportunities, Future Prospects
  3. Optimizing Gene-editing Experimental Designs for iPS Cells

Laboratory 1:

  • In Silico Design of guide RNAs (crRNA and sgRNA)
    • Optimized design of CRISPR/Cas9 systems for genome editing: analysis of representative cases

Laboratory 2:

  • Transfection of iPS cells with CRISPR/Cas9 reagents:
    • Part 1: Coat plates and make media
    • Part 2: Set-up Cas9 mRNA/crRNA:tracrRNA ribonucleoprotein complexes for transfection
    • Part 3: Harvest, replate and transfect iPSCs
    • Part 4: Re-plate and Transfect iPSC in suspension
  • Molecular synthesis of sgRNAs: annealing of crRNA encoding dsDNA oligonucleotide duplexes for cloning into pX330 vector
  • Molecular synthesis of sgRNAs: ligation of annealed DNA duplexes into pX330 vector and bacterial transformation

Lectures:

  1. Development of In Vitro Models via CRISPR Genome Editing
  2. Application of CRISPR/Cas9 for Genome Editing in Pluripotent Stem Cells

Laboratory 3: 

  • Review transfection efficiency by epifluorescence

Laboratory 4: 

  • Review transfection efficiency of iPSC cultures under epifluorescent microscope
  • Harvest transfected iPSCs and perform single-cell cloning

Laboratory 5: 

  • Discussion SURVEYOR (CEL-1) Mutation Detection Assay: Basic Principle and Workflow
    • CEL-1 Assay, Part1: Lyse iPSCs for Surveyor Genomic purification
    • Set-up the SURVEYOR PCR Reaction
    • Molecular synthesis of sgRNAs: Prepare T7-sgRNA amplicons for in vitro transcription
    • Molecular synthesis of sgRNAs: Setup the in vitro transcription reaction to prepare sgRNA
    • Calculate the efficiency of bacterial transfection and pick bacterial colonies for miniprep isolation of sgRNA-pX330 plasmid constructs
    • Prepare of Agarose Gels for Nucleic Acid Analyses

Lectures:

  1. Applications of dCas9 nucleases and disease modelling with CRISPR/Cas9 systems

  2. iPS cells in tissue engineering and other clinical applications

  3. iPSC technology to enable drug screening and preclinical evaluation

Laboratory 6: 

  • Assessment of pluripotency in iPSC cultures by live Tra-1-81 staining
  • CEL-1 Assay:
    • Part 2: Re-Annealing Step
    • Part 3: Digestion Step
    • Part 4: Agarose Gel Electrophoresis to determine the incidence and estimate frequency of indel mutagenesis
  • Analyze the Outcomes of PCR Test
  • Molecular synthesis of sgRNAs: Purify sgRNA and assess its quality by Tape Station and BioAnalyzer tests
  • Purification and agarose gel analysis of sgRNA-pX330 plasmid DNAs

Course Co-Director

Serguei Kozlov, PhD, MBA, PM

Dr. Kozlov received his Master of Science diploma in applied mathematics and theoretical physics from Moscow Institute of Physics and Technology in Russia, subsequently earning a PhD degree in molecular genetics and neurobiology from the Institute of Biochemistry, University of Zurich, Switzerland. He subsequently received a Master of Business Administration degree from Robert Smith School of Business, University of Maryland and also holds several other professional certifications and credentials.

Dr. Kozlov joined the National Cancer Institute in 1999 as a postdoctoral fellow in Cancer and Developmental Biology lab where he was promoted in 2003 to senior research fellow and subsequently in 2007 to the staff scientist appointment. In 2008 he joined SAIC Inc. (later renamed to Leidos BMR, Inc.) where he established Center for Advanced Preclinical Research – a translational resource providing interdisciplinary support and expertise in establishing next-generation animal cancer models, developing technologies for advanced manipulation of mammalian genomes for the purpose of preclinical modeling, and directing early stage drug development projects to accelerate clinical introduction of promising efficacious cancer therapeutics.

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Course Co-Director

James Kehler VMD, PhD is a comparative stem cell biologist who thrives on developing productive collaborations to translate scientific discoveries into transformative products. He trained at the University of Pennsylvania, where he received his VMD in 2002, and PhD in Cell and Molecular Biology in 2004. James has worked as a visiting researcher at the National Institutes of Health for over 10 years, where he and his collaborators at NEI, NCI, NINDS, NIDDK and NIAAA developed animal and stem cell-based models of human diseases. He has run workshops on reprogramming both at the NIH, as well as internationally. James has worked and consulted for several stem cell companies from product development and management to directing custom reprogramming and gene-editing services. James recently joined MTI-GlobalStem, as Director of Scientific Alliances to foster collaborative research projects with academic, biotech and pharmaceutical partners.

The Bioscience Education Center

Montgomery College
20200 Observation Drive
Germantown, MD 20876