Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a bacterial immune system that has been modified for genome engineering. This groundbreaking technology resulted in a Nobel Prize for Emmanuelle Charpentier and Jennifer Doudna in 2020. CRISPR consists of two components: a guide RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas9-binding (trRNA) and ~20 nucleotide spacer or targeting sequence which defines the genomic target to be modified (crRNA). Cas9 induces double-stranded breaks (DSB) within the target DNA. The resulting DSB is then repaired by either error-prone Non-Homologous End Joining (NHEJ) pathway or less efficient but high-fidelity Homology Directed Repair (HDR) pathway. The NHEJ pathway is the most active repair mechanism and it leads to small nucleotide insertions or deletions (indels) at the DSB site. This results in in-frame amino acid deletions, insertions or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. Ideally, the end result is a loss-of-function mutation within the targeted gene; however, the strength of the knockout phenotype for a given mutant cell is ultimately determined by the amount of residual gene function.

About This Material

This is a Hands-on Tutorial from the GTN which is usable either for individual self-study, or as a teaching material in a classroom.

Questions this will address

• What are the steps to process CRISPR screen data?
• How to identify essential genes across experimental conditions?

Learning Objectives

• Apply appropriate analysis and quality control steps for CRISPR screen data
• Identify differentially enriched genes across conditions
• Generate volcano plot to visualise results