Homo sapiens
Protocols: We induced a deletion of the regulatory region (chr14:21081182-21082486, 1.3 kbps GRCH37) in HEK293 cells using CRISPR/Cas9 genome editing and derived three independent clonal cell lines with homozygous deletions of the FMRE by culturing single genome-edited cells and screening the resulting colonies for FMRE deletions. For genome editing, single guide RNAs (sgRNAs) targeting the 5' and 3' boundaries of the FMRE were designed using the MIT CRISPR Designer software (http://crispr.mit.edu). The following sgRNAs were designed to induce 1,342 bp FMRE deletions (knockouts; KO) in HEK293 cell lines, resulting in FMRE-KO cell lines: 5'-CACCGTCATCACTCTATTGCTCCAC-3' (5' sgRNA) and 5'-CACCGTCGCCTTCCGCTTAATATGG-3' (3' sgRNA). The designed sgRNAs were ordered from Integrated DNA Technologies (IDT) as custom DNA oligonucleotides and cloned into the pSpCas9(BB)-2A-GFP plasmid (PX458, Addgene). Transfections were performed with Lipofectamine 3000 Reagent (Thermo Fisher Scientific) on HEK293 cells cultured in 6-well plates in DMEM medium supplemented with 10% (vol/vol) FBS at 37°C and 5% CO2. After 24 hours of incubation, the GFP+ fluorescent cells were isolated by FACS sorting and returned to incubator for further growth. Next, single cells were isolated by serial dilutions and cultured in 96-well plates at concentration of 0.5 cells per well. The colonies were inspected for clonal appearance after 1 week and expanded for additional 2-3 weeks. The clonal populations that reached ~90% confluency was subsequently split for DNA and RNA extractions. FMRE-KO clonal cell lines were identified by targeting the deleted genomic region and its boundaries with PCR experiments. Our FMRE-KO cell lines were grown from single cells, established and selected over at least five passages to enable our functional experiments. Therefore, no acute genotoxic stress would be expected as cells recover from genome editing experiments over passaging. Genomic DNA (gDNA) from wildtype and CRISPR/Cas9-edited clonal cell populations was extracted using DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer's instructions. The quantity and quality of gDNA was assessed with NanoDrop ND-2000 (Thermo Scientific). FMRE-KO cell lines were identified by two subsequent PCR experiments. We amplified 100-200 ng of gDNA using recombinant Taq DNA polymerase with 1X PCR buffer and 1.5mM MgCl (Invitrogen), and 500 nM of each primer (PCR-1: F1 5'-TCTGTTGCCAGGAAGGGATA-3', and R1 5'-AGGAAAAAGCCGTGGAAAAT-3'; wildtype band 1,564 bps, knockout band 222 bps; PCR-2: F1, and R2 5'-GGGATGTGAAGAAGCAAAGC-3', wildtype or band 555 bps, no detectable PCR product in knockout). The homozygous genotype of these cell lines was confirmed using Sanger sequencing (ACGT Corp) on PCR products (222 bps) amplified from the gDNA spanning the target sites of sgRNAs. Total RNA extraction was carried out using RNeasy Plus Mini kit (Qiagen) according to manufacturer's instructions. Extracted RNA was quantified using the Qubit fluorometer (Life Technologies) and quality was assessed using the Tapestation 2200 device (Agilent). RNA integrity number (RIN) was obtained for each RNA sample and indicated high quality (RIN range 9.4 to 9.8). High-throughput RNA sequencing (RNA-seq) was performed on three HEK293 FMRE-KO clonal cell lines and three wildtype HEK293 cell lines of different passages. Sequencing was completed at the core genomics facility of Ontario Institute for Cancer Research (OICR) using standard protocols. RNA-seq libraries for wildtype HEK293 cell lines and FMRE-KO cell lines were constructed using the KAPA Hyper RNA kit (Roche), quantified using the Fragment Analyzer device (Advanced Analytical), and pooled according to concentrations determined by qPCR.