FIG 5.

Cas9-based in situ tagging of the essential genes in M. maripaludis. (A and D) Schematics showing the in situ tagging at two genes encoding the RNA polymerase subunit L (RpoL) (A) and the transcription termination factor aCPSF1 (D). The pMEV4 plasmid was used to express sgRNA targeting the C terminus of RpoL or aCPSF1 (orange blocks); the donor sequences contain RpoL and aCPSF1 with the C terminus fused with His₆-3×Flag tag (blue block). The PAM nucleotides are shown in red letters. (B and E) Five randomly selected transformants were screened using PCR with chromosome-specific primers F5/R5 or F6/R6 as depicted in panels A and D. Black and red arrows indicate the PCR products amplified from the wild-type and mutated genomes, respectively. M, dsDNA size marker. (C and F) DNA sequences of representative PCR products amplified using F5/R5 and F6/R6, depicted in panels A and D, from the 5 mutants.