Saturation_Genome_Editing_of_BAP1

Saturation Genome Editing of BRCA1 Associated Protein 1 (BAP1). This study aims to functionally characterize saturating-levels of mutation at the BAP1 locus. We will target the Coding Sequence (CDS), splice-site containing intron and 3'UTR of the transcript: ENST00000460680.6. CRISPR-Cas9 and variant harbouring dsDNA repair templates will be co-transfected into HAP1 cells (Lig4-& clonal Cas9 expressing, sorted for 1n ploidy) to edit populations of cells. Duplicate libraries will be used to target most regions (that is the same oligonucleotide library composition for a region, but with a different sgRNA used for the CRISPR-Cas9 component). Targeted regions will then be sampled with gDNA extracted from cells at day 4, 7, 11, 14 & 21 after transfection. Amplicons are then generated for each of these time points in triplicate. Amplicons are composed of edited BAP1 locus. Oligonucleotide libraries were generated using the software package VaLiAnT (https://github.com/cancerit/VaLiAnT) and synthesized by Twist Bioscience. Libraries contain SNVs, in-frame deletions, an alanine and stop-codon scan, 1 base-pair deletions and tandem deletions in exon flanking intron. Clinical and population-observed mutations were also incorporated into edited regions (or 'targetons') by including accessions in ClinVar release: 20200927 and gnomAD release: r3.0. Libraries can be accessed here: https://gitlab.internal.sanger.ac.uk/team113_sge/team113_sge_libraries/bap1. After gDNA and PCR sampling (to enrich for the edited locus). Amplicons will be processed to add Illumina primary adapters and indexes and pooled. Sequencing will be performed on Illumina platforms. HISeq2500 and 500 SE reads (dual indexed pools) will be standard. The amplicons are roughly 245-300bp in length. Library sizes are roughly 1000 variants for each amplicon. We will aim for 500x coverage, which equates to roughly 500,000 reads allocated per library. Fastq files will be processed through a pipeline to obtain read-counts for variants with subsequent down-stream analyses performed to calculate the depletion kinetics of the variants within target regions.