Fastdigest drai

sgRNAs were designed with the CRISPR/Cas9 target online predictor tool CCTop with standard parameters (PAM = NGG, target site length = 20 nt, core length = 12 nt, max mismatches = 4, mouse genome = GRCm38/mm10) (24 (link)). The specific target sites were selected to induce DSBs in the intronic 5′ and 3′ regions of the target exon. The sgRNA target sites on the donor were replaced by the nucleotide sequences of the loxP sites to prevent CRISPR/Cas9 mediated cutting of the dsDNA donor. The generation of sgRNA constructs and in vitro transcription were carried out according to the protocol of Stemmer et al., 2015 (24 (link)). Purchased sgRNA oligos were annealed and cloned into the DR274 vector (25 (link)) (DR274 was a gift from Keith Joung, Addgene plasmid #42250) via the BsaI restriction site. The resulting sgRNA plasmid was linearized with FastDigest DraI (Thermo Scientific) and served as a template for sgRNA in vitro transcription using the T7 MEGAshortscript Kit (Thermo Scientific, AM1354) according to the manufacturer's protocol. sgRNAs were purified using the RNeasy Kit (Qiagen, 74104). For quality control, 450 ng of sgRNA was mixed with 2x RNA loading buffer and loaded onto a 1.5% TAE gel. sgRNA integrity was confirmed by the presence of a band at the 200 bp position of the DNA marker.

Antibodies: anti-MGME1 antibody (Sigma, HPA040913), rabbit-anti-vinculin (Cell Signaling, #13901), goat-anti-rabbit lgG HRP (Antibodies online, ABIN101744).
Recombinant constructs: CAG-Cas9-T2A-EGFP-ires-puro (Addgene # 78311).
Enzymes: FastDigest EcoRV (Thermo Scientific FD0303), FastDigest DraI (Thermo Scientific FD0224), FastDigest BamHI (Thermo Scientific FD0054), FastDigest MssI (Thermo Scientific FD1344), FastDigest Alw44I (Thermo Scientific FD0044), TopoIV (Inspiralis T4001), RNaseI (Thermo Scientific EN0601), T4 ligase and NEBNext Quick Ligation Reaction Buffer (New England Biolabs B6058).
Kits: High Prime DNA Labeling Kit (Roche 11585584001), NEBNext FFPE DNA Repair Mix (New England Biolabs #M6630), NEBNext Ultra II End repair/dA-tailing Module (New England Biolabs #E7546), Blunt/TA Ligase Master Mix (New England Biolabs M0367).