RNA-seq of human beta-cell line EndoC-betaH1 with and without single nucleotide replacement in the human TIA1 gene (E384K mutation)

Single nucleotide replacement in the human TIA1 gene (E384K) in human EndoC-βH1 cells was achieved by PE according to a previous publication (Anzalone, Randolph, Davis et al., 2019). CRISPR PE guide RNA (pegRNA) and nicking sgRNA (ngRNA) sequences were designed using the web resources available at https://drugthatgene.pinellolab.partners.org/primevar. PE plasmid is pCMV-PE2-P2A-GFP (Addgene plasmid #132776) expressing Cas9 H840A with co-translational GFP expression. PegRNA plasmid was constructed by ligation of annealed oligonucleotides including sequences of spacer, scaffold, and 3' extension into BsaI-digested pU6-pegRNA-GG-acceptor (Addgene plasmid # 132777). Plasmids expressing ngRNAs were constructed by ligation of annealed oligonucleotides into U6-BsmBIcassette-Sp-sgRNA BPK1520 (Addgene plasmid # 65777) (Kleinstiver, Prew, Tsai et al., 2015). EndoC-βH1 cells were seeded in Geltrex Matrix coated T25 flasks. Cells were transfected at approximately 60% confluency with 5 uL lipofectamine 2000 (Thermo Fisher Scientific) together with 3.6 µg PE plasmid, 1.2 µg pegRNA plasmid, and 0.4 ug ngRNA plasmid according to the manufacturer’s instructions. Cells were cultured for three days followed by sorting of GFP-positive cells (approximately 10%) by flow cytometry. GFP-positive cells were seeded in Geltrex-coated 6-well plates at various cell densities, and cultured for another five weeks. At intermediate cell densities, individual colonies developed (five wildtype (WT 1-5 and three mutated (Mut 1-3) that were picked and re-seeded into a 96-well plate for seven days followed by reseeding into two wells of 96-well plates for each clone. One well of the cells from an individual clone was used for DNA isolation (Qiagen) and PCR amplification of the amplicon covering the TIA1 mutation site. The PCR product was then sequenced at Eurofins Genomics for detection and conformation of the homozygous point mutation (G>A on the sense strand, E384K).