sgRNA Design

Guide RNAs (sgRNAs) were designed using CRISPick GPP sgRNA designer from the Broad Institute (Doench et al., 2016; Sanson et al., 2018; https://portals.broadinstitute.org/gppx/crispick/public) by providing DNA sequence for the region spanning the distal poly(A) site. In the case of Mprip, each output sgRNA was inspected assuring that one sgRNA targets upstream of the dPAS and one downstream, giving preference to higher “pick order” and “combined ranked sequences”. sgRNA oligos used for Mprip long 3′ UTR isoform (Mprip-L) knockout are found in Supplementary Table 2.

For bulk sgRNA analysis, the same CRISPick GPP sgRNA designer tool was used in bulk mode. Genomic coordinate bed file for the 150 bp upstream of the most distal 3′ UTR end was generated using the QAPA dPAU file as a reference. Bed file for the 150 bp downstream region was generated in a similar way for each of the genes. Then DNA sequences were extracted in fasta format using bedtools getfasta and GRCm38 mouse genome. Upstream and downstream fasta sequences were separately inputted for sgRNA design 500 sequences at a time. Only the top rank sgRNA was used for further analysis. Upstream and downstream sgRNA cut position information was used to estimate the expected deletion size for each gene. Data processing and plotting was performed in R.