Illustration from Scientific Research

The S. rosetta cRFXa locus encodes a protein that contains an N-terminal DNA-binding domain (DBD) followed by two conserved domains of unknown function (B and C) and a dimerization domain (DIM). The cRFXa locus was targeted by a guide RNA (gRNA) that anneals to an exon near the 5' end of the gene coupled with a homology-directed repair template that inserts a cassette (TTTATTAATTAAATAAA) that encodes an early stop codon (*in translation product, gray-shaded letters). The edited allele is called cRFXaPTS (for premature termination signal) and codes for a truncated polypeptide of 24 amino acids (see Data S1, File S4 for genome editing information). Two independent cRFXaPTS mutants, cRFXaPTS-1 and cRFXaPTS-2, were recovered. The cRFXaPTS-1 strain was reverted to a wild-type polypeptide sequence to create the cRFXaREV strain, which harbors a synonymous GTC -> GTG (Valine) that allows its genotype to be distinguished from that of cRFXaWT cells. DSB, double-strand break; PAM, protospacer adjacent motif. See Figure S2A for genotyping confirmation.

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