Foundations of CRISPR/Cas9 Technology 

I. Basic Principles of Genome Editing Technologies: From ZNF to CRISPR

II. 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

III. Structural Aspects of CRISPR/Cas9 Target Recognition and Biologic Patterns Among the Family of Bacterial Targeted Endonucleases

IV. Modified CRISPR/Cas9 and Non-Cas9 Systems for Gene Editing

V. CRISPR vs. RNAi Technologies: Strengths, Weaknesses, Opportunities, Future Prospects

Laboratory and Discussion:

• Conceptualization of CRISPR/Cas9 Mutagenesis Project: Strategies, Resources, Pitfalls

• Experimental Design for Successful CRISPR/Cas9 Project (Examples of p53 and Brca1 Conditional Allele Engineering and Exon Swap in mMSLN Locus)

• Planning, Construction, Synthesis, and Quality Control of CRISPR/Cas9 Nucleic Acid Reagents:

  - Design and Preparation of Oligonucleotides

  - Annealing ssDNA Oligos into dsDNA Duplex for Cloning

• Cloning of dsDNA Oligonucleotides into Vector for sgRNA Production

• Genomic DNA Purification from ES and Mouse Tail Biopsies: Sample Lysis

• Cell Transfection with CRISPR Reagents Using Nucleofection Technology: An Overview

• Preparation of ES (Bruce 4 line) and PancO2 Cells for Nucleofection

Applications of CRISPR/Cas9 Technology

I. Development of In Vitro Models via CRISPR Genome Editing

II. Molecular Design of CRISPR Libraries and Their Applications for Functional Screening and Gene Discovery

III. Modifications of CRISPR Technology Using dCas9 Endonuclease: Mechanisms and Applications

IV. Application of CRISPR/Cas9 for Genome Editing in Pluripotent Stem Cells

V. Accelerated Engineering of Novel Mouse Models with CRISPR/Cas9

Laboratory and Discussion:

• Review of Available Bioinformatics Resources to Identify and Design Efficient sgRNA Options

• Practical Exercise on Target Site Selection and Design of sgRNA (using mouse PTEN gene as a test case)

• Plasmid DNA Isolation for Individual sgRNA Clones and Verification of Their Identity by Restriction Digest and Agarose Gel Electrophoresis

• Gene Editing via HDR: Aspects of Donor DNA Design and Tips for Assuring the Efficient Recombination

• SURVEYOR (CEL-1) Mutation Detection Assay: Basic Principle and Workflow

  • CEL-1 Assay, Part1: Purification of Genomic DNA and Setting the PCR Reaction

  • CEL-1 Assay, Part2: Re-Annealing Step

  • CEL-1 Assay, Part3: Digestion
• Nucleofection of ES and PancO2 Cells

Advanced Concepts of Genome Editing Using CRISPR in Translational Research and Clinical Practices

I. Antiviral Properties and Applications of Targeted Endonucleases

II. Building Murine Models of Disease for Translational Research and Preclinical Applications by Precision Gene Editing

III. Emerging Clinical Applications of CRISPR/Cas9 as Promising Strategies in Gene Therapy and Disease Correction

IV. Facilitating Advanced Drug Development and Evaluation Using CRISPR/Cas9

Laboratory and Discussion:

• PCR Amplification of sgRNA Segment and Setting the Transcription Reaction

• CEL-1 Assay, Part4: Agarose Gel Electrophoresis to Analyze the Outcomes of PCR Test

• Genotyping of Genetically Engineered Mouse Models by PCR, Southern Blotting and DNA Sequencing: Basic Principles

• Practical Exercise: Identifying Various Allelic Combinations in Conditionally Targeted mDuox2 Locus by PCR Assay on Mouse Tail Genomic DNA

• Analysis and Interpretation of Genotyping Experiment Outcomes

• Concluding Part: Debriefing of All Experimental Results, Analysis of Pitfalls, and Discussing Possible Next Steps

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.