Fig. 4

Disruptions of two uncharacterized genes, Bip1 and Bip2, with Mo_tRNA^Arg-gRNA-Cas9/pKOV21 system in P. oryzae. a Strategy for Bip1 gene replacement, including the sgRNA-guided Cas9-induced DSB site on Bip1. H indicates Hind III used to digest genomic DNAs for Southern blot. The probe used for Southern blot was labeled. b Ten Neo-sensitive transformants were checked by PCR with the Hyg_up/check_up and Hyg_down/check_down primers (as shown in a) to verify Bip1 replaced by Hyg correctly. c PCR detection of Cas9 insertion in genomes of the ten transformants mentioned in b. d Δbip1 mutants verified by Southern blot. e Strategy for Bip2 gene replacement, B indicates Bgl II used to digest genomic DNAs for Southern blot. The probe used for Southern blot was labeled. f Four Neo-sensitive transformants were checked by PCR to verify correct replacement of Bip2 by Hyg. g Southern blot for verifying Δbip2 mutants. h PCR detection for Cas9 insertion in genomes of the transformants in f