Understanding the molecular mechanisms underlying adaptation is a central goal in modern evolutionary biology. The CRISPR-Cas9 system is a powerful genome editing tool that can be used for evolutionary studies, providing a reliable way to functionally validate candidate genes in emerging model species. In this workshop, students will learn the basics of designing and performing CRISPR gene knockout experiments aiming to connect genotype and phenotype. The applications of CRISPR technology in evolutionary studies will be illustrated, and lectures will focus on the theoretical framework for a successful CRISPR-based genome editing experiment. This workshop will include both fundamental theoretical lessons and hands-on sessions with practical examples of genomic data analysis. All data used in this workshop has been chosen from up-to-date scientific literature. This workshop will focus on experiment design, selection of methods and data analysis. General “starting-point” bench protocols will be shared, and each day will include a “Discussion and Q&A” session where participants can fine-tune their own projects with the instructors.

The workshop is intended for students (undergraduate and graduate), researchers, and technicians in the biological sciences interested in implementing the CRISPR-Cas9 tool in their own projects. A basic background in biology, specifically in genetics and molecular biology, is required. The examples used in this workshop will focus on the application of CRISPR in evolution and ecology studies, mainly using examples from– but not restricted to- the study of insects. Therefore, participants are expected to understand the central role of nucleic-acids (i.e., DNA/RNA) in evolution. Furthermore, most hands-on sessions include sequencing data analysis, and students are expected to have a general knowledge of bioinformatics (e.g., BLAST homology-based search and multiple sequence alignment). There will be theoretical lectures and hands-on practical exercises using web-based tools, license-free software, and UNIX-based command line programs. Experience using command-line based programs will be advantageous, but not required. An introduction to UNIX commands will be provided.

•    Understand the fundamentals of the CRISPR-Cas9 genome-editing tool.
•    Design a reliable CRISPR-based gene knockout experiment.
•    Annotate a gene of interest in a non-model species.
•    Design and evaluate species-specific single guide RNAs (sgRNAs).
•    Efficiently synthesize sgRNA and assemble optimized Cas9/sgRNA mixes.
•    Screen for G0 CRISPR mutants using a fast genotyping method.
•    Design genotyping strategies according to project needs.
•    Analyze Cas9-induced genomic variants by SANGER and Illumina deep sequencing.

Daily schedule: from 2 – 7 pm Berlin time.

Day 1.
•    Introduction to CRISPR-Cas9 technology
•    Applications of CRISPR in evolution and ecology studies
•    Design considerations of CRISPR-based gene knockout experiments
•    Introduction to basic UNIX commands
•    Manual gene annotation
•    Discussion and Q&A

Day 2.  
•    Design considerations of optimal sgRNAs
•    Design custom target-specific sgRNAs
•    Analysis of sgRNA oligos
•    Synthesis of sgRNAs by in vitro transcription
•    Methods for evaluation of sgRNA efficiency
•    Discussion and Q&A (time-dependent; group discussion)

Day 3.
•    Delivery of CRISPR-Cas9 components
o    (TBC) Video session: Microinjections in Medfly
o    (TBC) Video session: Microinjections in Heliconius butterflies
•    Screening strategies to identify Cas9-induced mutations
•    Genotyping CRISPR-modified individuals
•    Analysis of deep sequencing data from G0 mosaic mutants (part 1)
•    Discussion and Q&A (time-dependent; group discussion)

Day 4.
•    Analysis of deep sequencing data from G0 mosaic mutants (part 2)
•    Considerations for development of biallelic mutant strains
•    Genotyping of mutant lines using long-PCR and WGS
o    Hands-on: SANGER sequencing analysis of developed mutant lines
Final considerations, discussion and Q&A

Cancellation Policy:

> 30  days before the start date = 30% cancellation fee

< 30 days before the start date= No Refund.

Physalia-courses cannot be held responsible for any travel fees, accommodation or other expenses incurred to you as a result of the cancellation.