Simply cut out - Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion in Trichoderma atroviride

Abstract

Trichoderma atroviride is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. However, genetic manipulation in this organism remains challenging due to the limited availability of versatile and efficient molecular tools. Here, we present a CRISPR/Cas9-based method for targeted gene manipulation using ribonucleoprotein (RNP) complexes combined with a transiently stable telomere vector. We successfully inactivated three genes-pks4 (spore pigment production), pyr4 (pyrimidine biosynthesis), and pex5 (peroxisomal matrix protein import receptor)-to demonstrate the system's utility. Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene inactivations in our case were achieved via non-homologous end joining (NHEJ), by co-transforming the transiently stable telomere vector carrying the hygromycin-resistance gene (hph), which was rapidly lost under non-selective conditions. This strategy enables marker-free genetic manipulation, supports vector recycling, and simplifies successive transformations. Overall, our method expands the genetic toolbox for T. atroviride, offering a fast and reliable approach for reverse genetics in this agriculturally important fungus.

Keywords: CRISPR/Cas9; Trichoderma atroviride; marker-free; telomeric vector; transient resistance.

FIGURE 1

Inactivation of the pyr4 gene in T. atroviride P1. (A) Schematic diagram of the pyr4 coding sequence on chromosome 1 with the Cas9 target site indicated scissors. Primers P1 (pyr4_1.1) and P2 (pyr4_2.1) were used for locus amplification. (B) Genotyping of eleven selected 5-FOA resistant and uracil/uridine auxotrophic transformants and T. atroviride wt. (C) Amplicon sequencing results covering a part of the pyr4 locus of the wt and eleven selected transformants. The corresponding amino acid sequence is given above the genomic sequence. The region targeted by the gRNA is underlined, the PAM sequence is framed in grey and the Cas9 cutting site is marked with a triangle. Asterisks (**) indicate the integration of sequences from different chromosomal regions. DEL (highlighted in red) indicates mutants with deletions in the py4 locus; INS (highlighted in green) indicates mutants with insertions. (D) Comparative assessment of growth of the wt and four pyr4 mutants on either PDA or PDA supplemented with 5 mM uracil.

FIGURE 2

Deletion of pks4 through a bipartite marker-free editing approach. (A) Schematic diagram of the pks4 coding sequence in the subtelomeric region of chromosome 5 with the Cas9 target sites indicated by scissors. (B) Absence of conidial pigmentation being indicative of pks4 gene inactivation in transformants 1, 2 and 3 (T1, T2, T3) upon growth on PDA.

FIGURE 3

Deletion of pex5 through a CRISPR/Cas9 assisted gene replacement approach in Δpyr4 background. (A) Schematic diagram of the pex5 coding sequence on chromosome 2 with the Cas9 target sites indicated by scissors. The coding sequence of N. crassa pyr4 (Nc pyr4), surrounded by its endogenous promoter (P) and terminator (T), was provided as repair template to replace pex5. Primers P1 (pex5_1.1), P2 (Nc_pyr4_2.3) and P3 (Nc_pyr4_1.3), P4 (pex5_2.1) were used for locus amplification. Pex5 is replaced by the N. crassa pyr4 gene. (B) Genotyping of ten transformants using primer pair P1, P2 (top) or P3, P4 (bottom). (C) Comparative assessment of growth of the wt and transformant 3 (T3) on either TMM or TMM supplemented with 1 μg/mL biotin.

FIGURE 4

Deletion of pex5 through CRISPR/Cas9-mediated excision. (A) Schematic diagram of the pex5 coding sequence on chromosome 2 with the Cas9 target sites indicated by scissors. Primers P1 (pex5_1.1) and P2 (pex5_2.1) were used for locus amplification. Excision of the pex5 coding sequence is followed by non-homologous end-joining (NHEJ), resulting in Δpex5 mutants. (B) Genotyping of eight transformants and T. atroviride wild type (wt). (C) Amplicon sequencing results covering part of the pex5 locus of the wt and transformants 3, 4, 6 and 8 (T3, T4, T6, T8). The region targeted by the gRNA is underlined, the PAM sequence is framed in grey and the Cas9 cutting site is marked with a triangle. The pex5 open reading frame (ORF) is enclosed in a rectangle. A DNA fragment of 1930 bp was excised. (D) Comparative assessment of growth of the wt and transformant 3 on either TMM or TMM supplemented with 1 μg/mL biotin.

FIGURE 5

Schematic representation of the different approaches followed in this study. Three different genomic loci were targeted: i) pyr4, ii) pks4 and iii) pex5. Inactivation of pyr4 is known to result in an uracil/uridine auxotrophic mutant, that can subsequently be utilized as recipient strain for transformation with a functional pyr4 sequence. For pyr4 inactivation, the efficacy of a single RNP without repair template was tested. pks4 disruption results in fungal mutants with spores lacking the green pigmentation. To delete pks4, a different approach utilizing two RNPs for complete excision of the coding sequence was tested, thereby employing the telomeric vector pTel-hyg for selection. To compare the two selection approaches, pex5 was selected as target. For pex5 deletion, two approaches were followed: (a) the pyr4 deficient strain was used as recipient, in order to test the efficiency of homology-directed repair (HDR) providing a functional pyr4 sequence from N. crassa. (b) The wt was transformed without repair template to verify the efficiency of non-homologous end-joining (NHEJ) as well as of the loss of the recyclable marker. Positive and negative points for each strategy are indicated on the right.