ENAGenomicsSCWellcome Sanger Institutehttps://www.ebi.ac.uk/ena/browser/view/PRJEB75748Saturation genome editing of the NUDCD3 gene. We aim to target the coding sequence (CDS) and splice-site containing intron of the transcript ENST00000355451.8. Guide RNAs and variant harbouring dsDNA repair templates will be co-transfected into HAP1 cells (Lig4 and clonal Cas9 expressing cells, sorted for 1n ploidy) to edit population of cells. Targeted regions will be sampled with gDNA extracted from cells at different time points (day 4, day7 and day15) after transfection. Amplicons will be generated in triplicate for each time point. The amplicons are composed of edited NUDCD3 locus. Oligonucleotide libraries are 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 will be also incorporated into edited regions (or 'targetons') by including accessions in ClinVar and gnomAD. 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 NovaSeq-SP. 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. Plasmid libraries will also be sequenced as part of this project. 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.ENAfalseSaturation_Genome_Editing_of_NUDCD3___Saturation_Genome_Editing_of_NUDCD3___2025-07-222025-07-22PRJEB75748