Fig. 1

Schematic overview of CRISPR–Cas12a- and CRISPR–Cas9-mediated genome editing in the thermophilic fungus M. thermophila. To edit the M. thermophila genome, either the Cas9 and sgRNA-expressing cassettes or Cas12a and crRNA expression cassettes were introduced into the recipient protoplasts. For the CRISPR–Cas9 system, the single-guide RNA (sgRNA) contains crRNA and tracrRNA. As compared to Cas9, Cas12a is a single-RNA-guided nuclease that does not require a tracrRNA and the crRNA consists of 19-nt direct repeat and 23-nt guide. Cas12a has a T-rich PAM sequence located at the 5′ end of the protospacer and cleaves DNA distal from the PAM and generates staggered ends. Once Cas9 or Cas12a nuclease generates the sequence-specific double-stranded DNA break (DSB), non-homologous end-joining (NHEJ) or homology-directed repair (HDR) processes mediate DNA modification at the cleavage locus. NHEJ produced small random insertion or deletion (indels) cleaved site, whereas HDR uses DNA donor template for the precise recombination. DR, 19 nt direct repeat; U6p, MtU6 promoter; Ptef1, Tef1 promoter; NLS, nuclear localization signal; TtrpC, trpC Terminator. TTTTTT, poly-T sequence used as terminator