Fig. 3

Deletion of the LpAsp and LpCht genes by CRISPR. Schematic representation of WT and deleted KO alleles of the LpAsp (a) and LpCht (b) genes generated using CRISPR/Cas9-induced homology-directed repair. 5’and 3’-UTRs, open reading frames and Hph gene are depicted in blue, green, beige, and red, respectively. The expected sizes of the PCR products for detecting the WT and KO alleles (not to scale) are also displayed for each gene. Genomic DNAs from WT L. passim (+/+), L. passim heterozygous (+/–) and L. passim homozygous (-/-) mutants of LpAsp and LpCht were analyzed by PCR to detect 5’WT, 5’KO, 3’WT and 3’KO alleles. Sizes of the DNA molecular weight markers are provided on the left. c Detection of LpAsp and LpGAPDH mRNAs in LpAsp heterozygous ( +/–) and homozygous (-/-) mutants, along with WT L. passim (+/+) through reverse transcription-PCR. A forward primer corresponding to the L. passim splice leader sequence was utilized. Sizes of DNA molecular weight markers are shown on the left. d Detection of LpCht and LpGAPDH mRNAs in LpCht heterozygous (+/–) and homozygous (-/-) mutants and in WT L. passim (+/+), by reverse transcription-PCR. Sizes of the DNA molecular weight markers are indicated on the left. CRISPR, Clustered regularly interspaced short palindromic repeats; hph, hygromycin resistance gene; KO, deleted (knockout); LpAsp, L. passim aspartyl protease; LpCht, L. passim chitinase; LpGAPDH, L. passim glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; WT, wild type; UTR, untranslated region