Double_CRISPR_knock_out_screen_of_ultraconserved_elements. Double_CRISPR_knock_out_screen_of_ultraconserved_elements

Across the human genome, there are 481 elements with lengths greater than 200 base pairs showing 100% conservation with mouse and rats, and >95% sequence conservation when compared to other vertebrates. These elements are distributed genome-wide, but surprisingly many are in non-coding regions. Some of these ultraconserved regions (UCEs) have been shown to act as enhancers during embryonic development but for many their function remains unclear. In this project we aim to perform a double CRISPR knock-out screen to study the functional effect of UCE deletion in cultured cell lines with cell fitness being used as a phenotypic readout. To expand our analysis, we have also identified a further 9893 sequence elements, where every position in the human genome has been aligned and compared with 240 additional mammalian species (the Zoonomian project) defining regions showing high conservation and thus regulatory/functional potential. From this large dataset, we have designed a CRISPR screen where two single guide RNA will simultaneously cut and delete a region of interest removing this sequence from the genome. Downstream studies will deploy saturation genome editing to define the nucleotides with regulatory potential in those regions found to influence cell growth/death.