Sox9^em1Rlb

The core region (TESCO) of the TES regulatory region 10-13 kb upstream of the gene was targeted with a pair of single guide RNAs (sgRNAs) using the CRISPR/Cas9 system. The proximal sgRNA (5'-caccgTCTGTTAACGTATGACACGA-3'; recognition sequence in capital letters) was targeted to sequence 8 bp downstream of the 3'. The distal sgRNA (caccGTTGGAGTTCCGATTTAGA) was targeted to sequence 41 bp downstream of the 5' end of the region (i.e. inside the region). Two stable lines were established that carried the exact 1260 bp deletion between the Cas9 cleavage sites. This 1260 bp deletion deletes 1252 of the 1293 bp TESCO sequence. One line also carried an 11 bp insertion of unrecognized sequence at the cleavage site. Only one of the lines was used for the experiments. (J:238708)