Fig 1.
Components of the CRISPR-Cas9 system.
Individual components in both panels are not drawn to scale. A) RNA guided nuclease Cas9 specifically cleaves a genomic target sequence. The protospacer and scaffold sections of the sgRNA are represented in light green and green, respectively. The protospacer basepairs with the complementary strand of the target sequence to form a D-loop. Note that efficient cleavage depends on the presence of the three bp PAM sequence in the target sequence, which is located directly downstream of the region invaded by the protospacer. Blunt-end cleavage occurs between bases located 3–5 bp upstream of the PAM, as indicated by scissors B) Liberation of the sgRNA, represented in light green/green, from a polymerase II transcript by intrinsic hammerhead (HH) ribozyme and hepatitis delta virus ribozymes (HDV) ribozyme represented in red/blue and orange respectively. Cleavage points are indicated by scissors. The blue part of HH basepairs with the protospacer for efficient cleavage. C) A fungal AMA1 based vector harboring Cas9 and sgRNA encoding genes are transformed into a fungus. The Cas9/sgRNA riboprotein induces a site specific DNA DSB. In the absence of a homologous template for HR repair (left lane), the DNA DSB is repaired by NHEJ; and error/prone repair by NHEJ results in mutations as indicated by yellow base pairs. In the presence of a linear or circular gene targeting substrate (right lane), specific Cas9 induced DNA DSBs may be repaired by HR resulting in a gene targeting event illustrated as insertion of an orange marker gene in this example.
Fig 2.
Construction of new CRISPR-Cas9 vectors for directed mutagenesis of filamentous fungi.
Construction of fungal CRISPR-Cas9 vectors with variable sgRNA genes controlled by gpdA promoter and trpC terminator (no DNA elements are drawn to scale). The vector backbone for construction of new Fungal vectors for Cas9 induced genetic engineering are derived from the plasmid series pFC330-333. Sticky ends for USER cloning are achieved by opening the PacI/Nt.BbvCI USER cassette of pFC330-333 by the concerted action of restriction enzymes PacI and Nt.BbvCI (left side of panel). The two PCR fragments necessary for construction of the sgRNA gene, are both obtained by using pFC334 as template (right side of panel). This vector contains a protospacer for targeting yA (in light green), which is represented by 20 Ns indicating that it is not intended to match the primer; and in principle could be any sequence. The sections of the sgRNA gene encoding the variable parts of the transcript, the 20 bases of the protospacer (in light green) and the reverse complementing 6 bases of HH (in light blue), are introduced via tails added to the ends of the two primers that define the down- and upstream ends of the two PCR fragments, respectively. The position of the resulting inverted repeat located in the variable regions is indicated by blue arrows (top of panel). After amplification, the two PCR fragments are fused and inserted into vector pFC330-333 (Four variants exist) by USER cloning in a single step. For this purpose, each PCR fragment is generated by primers containing a tail with a uracil base (in purple). Elimination of the uracil bases in the PCR fragments by Uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII (USER Enzyme) results in the production of pairwise complementary overhangs at the ends of all fragments allowing selected ends to be fused in a directional manner. For simplicity, all complementary ends are visualized in the same color.
Fig 3.
RNA guided Cas9 efficiently introduces directed mutations into the yA gene of A. nidulans.
A. nidulans transformed with A) pFC331 encoding Cas9, but not a sgRNA, and with B) pFC334 encoding Cas9 and a yA specific sgRNA. C) Stabs of green transformants on solid selective medium from the plate shown in panel B. D) Stabs of yellow transformants on solid non-selective media. Despite loss of pFC334 they remain phenotypically stable
Table 1.
Mutation spectrum of RNA guided Cas9 mutagenesis.
Fig 4.
RNA guided Cas9 efficiently introduces directed mutations into albA and pyrG of A. aculeatus.
A. aculeatus transformed with A) pFC332 encoding Cas9, but not a sgRNA plated on selective media. B) spores harvested from colonies shown in panel A, plated on medium containing 5-FOA, which impairs growth of pyrG wild-type strains preventing conidiation. C) pFC336 encoding Cas9 and an albA specific sgRNA. D) Spores harvested from transformants expressing Cas9 and pyrG specific sgRNA, pFC337, plated on medium containing 5-FOA. Note that colonies formed by pyrG mutant strains propagate normally and produce conidia.
Fig 5.
RNA guided induced DNA DSBs efficiently stimulate gene targeting in A. nidulans and A. aculeatus.
A. nidulans and A. aculeatus were co-transformed with a CRISPR-Cas9 plasmid in combination with a gene targeting substrate. yA of A. nidulans was targeted by a linear gene targeting substrate; and albA of A. aculeatus was targeted by a linear as well as by a circular gene targeting substrate as indicated to the left of panels. The presence of an sgRNA gene (yA specific for A. nidulans and albA for A. aculeatus) gene in the CRISPR-Cas9-vector is indicated above panels. Selection conditions for each experiment are indicated above panels. Single selection refers to selection for the gene substrate alone; and double selection refers to selection for both the gene targeting substrate and the CRISPR-Cas9 vector.
Table 2.
Strains used in this study to implement functional CRISPR.
Table 3.
Protospacers used in this study.