Fig. 1.

Gene editing strategy and construction of B. bassiana (ΔBbsmr1) mutants. A) Gene edition strategy. Red arrow corresponds to the specific Cas9 target site. Black arrows correspond to diagnostic primers. B) Diagnostic PCR for detection of positive mutants (ΔBbsmr1) using specific markers (long read primers: Bbsmr1-F + Bbsmr1-R) before monosporic purification step in 1% agarose gel. The PCR products expected band sizes were 1.9 kb and 4.8 kb for the WT strain and the putative transformants (containing the insert DNA of geneticin resistance gene), respectively. C) Diagnostic PCR for site-specific mutation in Bbsmr1 using specific markers (short read primers: Bbsmr1-F + Gent_R) in 1% agarose gel to confirm the positive transformants. The ~ 2.4 kb band corresponds to a positive ΔBbsmr1 mutant, confirming the integration of geneticin cassette into Bbsmr1 gene. As expected, no band appears in WT due to the absence of the geneticin resistance gene. All homokaryon mutants are highlighted with a green inverted triangle and they show a band in both gels (B, C). Ctrl is the negative control without any DNA, and 1 kb marker was used in the the gels