alias: KO1
title: H9_53BP1KO1
design: To generate lentiviral constructs, exon-specific gRNAs were designed using the clustered regularly interspaced short palindromic repeats (CRISPR) Design Tool (http://crispr.mit.edu/) to minimize offtarget effects. Oligos (Integrated DNA Technologies) were synthesized, annealed, and cloned into vector LentiCRISPRv2 (Addgene). gRNA targeting the firefly luciferase gene (exons 6 and 7) was used as non-specific control. Viral constructs were co-transfected with VSVL, REV, and HDL into 293T cells by using Lipofectamin 3000 (Life Technologies) to generate lentiviruses. Lentiviral particles were harvested in mTeSR1, and titer was quantified by the Lenti-X qRT-PCR titration kit (Clontech). For lentiviral transduction, ESCs were inoculated 2 consecutive times with lentiviral particles (3 h for each time) and allowed to recover in mTeSR overnight. Puromycin (0.375 g/mL; ThermoFisher Scientific) selection started approximately 24 h after transduction. Single cells from transduced and selected populations were sorted into 96-well plates (BD FACSAria  Fusion) and amplified. Genomic DNA from single-cell clones was harvested by PureLink  Genomic DNA mini kit (Life Technologies). To assess potential DNA indels, genomic DNA was amplified using exon-specific PCR primers. PCR products were cloned into pJET vector (Life Technologies) and sequenced by the Sanger Sequencing Core facility at St. Jude. Supplementary Tables 5 and 6 list primers used for this assay.
identifiers.namespace: SUB3337190
identifiers.id: KO1