August 11-13, 2021

Wednesday-Friday

9:00am-5:00pm

1 Hour Lunch Break

895

Workshop Fee

This rigorous three day live remote 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.

remote learning
Real-Time Interactive Lecture, Laboratory Protocol and Discussion
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Team taught by active researchers
Reference Materials Included
Lectures, Lab Protocols, Reference Materials included
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Space limited to 24 participants
Save Money and Time
No Travel, Convenient and Cost Effective

Course Co-Director

Course Director

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

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 Genomic Cleavage Detection Assay: Basic Principle and Workflow
    • Genomic Cleavage Detection Assay- Part1: Lyse iPSCs and set-up PCR Reaction

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
  • Genomic Cleavage Detection 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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Instructor

Dr. Jean-Phillippe Richard has over 12 years of experience in stem cell research, first as an NIH Fellow; then as a research associate at the Johns Hopkins School of Medicine; Department of Neurology, Ansari Stem Cell Core.  Currently Dr. Richard is the Stem Cell Director at REPROCELL, having dual experience in bench science (conceiving/executing experiments) and in designing/promoting/managing internal and external collaboration related to broad research initiatives.

The Bioscience Education Center

Montgomery College
20200 Observation Drive
Germantown, MD 20876