Understanding the molecular mechanisms underlying adaptation is a central goal in modern evolutionary biology. The CRISPR-Cas9 system is a powerful tool for evolutionary studies, offering a reliable way to investigate candidate genes in non-model species. In this course, students will learn the fundamentals of designing and performing CRISPR gene knockout experiments to connect genotype and phenotype. The applications of CRISPR technology in evolutionary studies will be illustrated, with lectures focusing on the theoretical framework for successful CRISPR-Cas9 experiments.

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.

* Instructors will use real-data examples from their projects and experiences in molecular entomology. However, the critical experimental design and molecular methods discussed in this course can be transferred to any other biological system with minor modifications. 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*

This online course will span five days and combine theoretical lessons with hands-on sessions and practical exercises. The focus will be on experiment design, method selection, and data analysis. Lectures will be delivered via live video conferences, while practical exercises will follow a flexible schedule. Hands-on activities using web-based tools will be conducted live, whereas exercises requiring command-line programs will be structured as follows: (1) live demonstration, (2) at-home reproduction (homework), and (3) next-day review and discussion. Each day will conclude with a discussion and Q&A session to wrap up daily content. A dedicated Slack workspace will facilitate class interactions and provide access to course materials. Special time will be reserved on the last day for one-on-one discussions, allowing participants to refine their projects with instructors' guidance. 

•    Understand the fundamentals of the CRISPR-Cas9 genome-editing tool.
•    Design a reliable CRISPR-based gene knockout experiment.
•    Functionally investigate a gene of interest in a non-model species.
•    Design and evaluate species-specific guide RNAs (gRNAs).
•    Efficiently synthesize gRNA and assemble optimized Cas9/gRNA mixes.
•    Screen for CRISPR-derived mutants using a number of genotyping methods.
•    Design genotyping strategies according to project needs.
•    Analyze Cas9-induced genomic variants by Illumina deep sequencing.
•    Critically interpret the results from a CRISPR-based KO experiment

Daily schedule: from 2 – 7 pm Berlin time.

Day 1.
•    Course welcome and introductions
•    Overview of the CRISPR-Cas9 technology
•    Applications of CRISPR-Cas9 system in evolution and ecology studies
•    Design considerations of CRISPR-based gene knockout experiments
•    Presentation to course hands-on project
•    (practical) Introduction to basic UNIX commands
•    Discussion and Q&A

Day 2.  
•    (practical) Manual gene annotation
•    Design considerations of optimal gRNAs
•    Synthesis of gRNAs by in vitro transcription
•    (practical) Analysis of gRNA oligos
•    Evaluation methods of gRNA efficiency
•    Discussion and Q&A

Day 3.
•    (practical) Design custom target-specific gRNAs
•    Delivery of CRISPR-Cas9 components: embryo microinjections
•    Screening for Cas9-induced mutations (in 2 parts)
•    Discussion and Q&A

Day 4.

•    Delivery of CRISPR-Cas9 components: DIPA and ReMOT
•    Genotyping CRISPR-modified individuals
•    (practical) Analysis of deep sequencing data from Cas9-derived mutants (in 2 parts)
•    Conclusion of the course hands-on project
•    Discussion and Q&A

Day 5

•     CRISPR-based control strategies against pest insects
•     Final considerations
•    One-on-one discussions (let’s talk about your project)

•    Course evaluation survey

Instructor

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.