 introduction to CRISPR screen analysis. Before diving into this slide deck, we recommend you to have a look at the following. What is CRISPR? What is a CRISPR screen? How is a guide RNA library created? What is the difference between a negative and positive screen? How to analyze CRISPR screen data? Describe what CRISPR screen data is. Outline how CRISPR screen data is generated and analyzed. CRISPR stands for clustered regularly into spaced short palindromic repeats. It's a bacterial immune system that has been modified for genome editing. It consists of two components, a guide RNA and a non-specific CRISPR associated endonucleus called Cas9. The guide RNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas9 binding and 20 nucleotide spacer sequence that binds to the genomic target. Cas9 induces a double stranded break within the target DNA. This results in in-frame amino acid deletions, insertions or frame shift mutations leading to premature stop codons within the targeted gene. With CRISPR knockout methods, ideally the end result is a loss of function mutation within the targeted gene. There are also CRISPR inhibition and activation methods, but in this tutorial we focus on knockout. The ease of generating guide RNAs makes CRISPR one of the most available genome editing technologies. It can be used for genome wide screens, enabling systematic targeting of tens of thousands of genes with one gene targeted per cell. With these screens we can identify the functions of genes such as those essential for cell survival, drug resistance or sensitivity. Pooled or arrayed screens can be performed. In this tutorial we analyze data from a pooled screen. Various guide RNA libraries are available and can be purchased. In this tutorial we use Bronello, which is a human genome wide CRISPR knockout library in a lentigide vector. Cells expressing the Cas9 enzyme are transduced with the guide RNAs at a low multiplicity of infection, MOI, aiming for a minimum starting representation of 300 for each guide, and Puromycin is used to remove cells without guides. CRISPR positive or negative screens can be performed. With a positive screen, few cells survive the treatment and we are interested in identifying genes whose guide RNAs increase are enriched, indicating knockout of those genes leads to resistance. With a negative screen, most cells survive after the treatment. In that case, we are interested in identifying genes whose guide RNAs decrease are depleted compared to a control for example vehicle, indicating knockout of those genes increases sensitivity to the treatment. In this tutorial we analyze data from a negative screen. Cells from the time points of interest are collected, genomic DNA is extracted, and the guide RNA region is amplified by PCR, followed by sequencing. This is an overview of the workflow used in the tutorial. We first repair the sequencing reads, checking quality and trimming adapters. We then use the magic toolkit to analyze, followed by visualizing results using volcano plots and identifying pathways. Thank you for watching.