CRISPR-Cas9 System Development for Myceliophthora thermophila

All primer sequences used in this study are listed in Additional file 7: Table S1. All PCR products were amplified using Phusion high-fidelity DNA polymerase (Thermo Fisher, Waltham, MA, USA). A codon-optimized Cas9 gene with attached hac-1 (MYCTH_2310995) nuclear localization signals (NLS-Cas9-NLS) was synthesized by Life Technologies (Invitrogen) for expression in M. thermophila and M. heterothallica. The synthetic NLS-Cas9-NLS, the strong constitutive tef1 (MYCTH_2298136) promoter of M. thermophila, and the TtprC terminator were amplified using paired primers (Additional file 7: Table S1). With the aid of a NEB Gibson assembly kit, these amplification products were assembled to form a Ptef1-Cas9-TtprC cassette (Additional file 8) and inserted into a p0380-bar plasmid [56 (link)] carrying the bar marker to generate the Cas9-expression vector p0380-bar-Ptef1-Cas9-TtprC. A Gibson assembly kit was also used to construct a Cas9-eGFP-expression vector (p0380-bar-Ptef1-Cas9-eGFP-TtprC) in which enhanced GFP (eGFP) was fused to Cas9 as a reporter gene (Additional file 8). To generate sgRNA expression plasmids, an sgRNA scaffold was synthesized by Life Technologies. To express sgRNA in protoplasts, the M. thermophila U6 promoter was amplified from ATCC 42464 genomic DNA using the primer pair U6-F/R (Additional file 7: Table S1) and then fused to the sgRNA scaffold fragment by fusion PCR using U6-F and gRNA-R (Additional file 7: Table S1). The resulting fusion fragment was cloned into a pJET1.2/blunt cloning vector to create the corresponding plasmid U6p-sgRNA (Additional file 9).
To select for specific sgRNAs targeting amdS (GenBank number: M16371.1), cre-1 (MYCTH_2310085), res-1 (MYCTH_2302052), gh1-1 (MYCTH_115968), and alp-1 (MYCTH_2303011), all sgRNA target sites in the genome of M. thermophila were identified using the sgRNACas9 tool [57 (link)]. sgRNA target sites with high scores were chosen and the corresponding oligos were ordered (Additional file 7: Table S1). All protospacer sequences used to target the five different genes are presented in Table 1. A target-directed M. thermophila U6 promoter-driven sgRNA was created by overlapping PCR with the primers given in Additional file 7: Table S1 and cloned into a pJET1.2/blunt cloning vector, which yielded the corresponding plasmids U6p-amdS-sgRNA, U6p-cre1-sgRNA, U6p-res1-sgRNA, U6p-gh1-1-sgRNA, and U6p-alp1-sgRNA (Additional file 9).