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Fig. 4 | Biotechnology for Biofuels

Fig. 4

From: Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila

Fig. 4

Schematic strategy of CRISPR–Cas-mediated marker recycling approach for iterative multiplex genome editing in M. thermophila. The hendecuple mutants were constructed in three successive transformations by alternatively using two selectable markers neo and bar. In the first transformation, a triple mutant was created by transient CRISPR–Cas12a/Cas9-mediated homology-directed repair (HDR). The neo cassette replaces an endogenous locus, and two target genes are both markerless deletions. In the second transformation, the insert of neo is removed and bar replaces a new endogenous locus in the triple-mutant host strain via HDR by the CRISPR–Cas12a/Cas9 system. In addition, two other gene loci undergo seamless gene replacement and markerless gene disruption. For the third transformation, the octuple mutant is selected for genetic manipulation using the CRISPR system. The selectable marker neo is inserted into a new endogenous locus, and the marker bar is scarlessly removed and the other new gene undergoes markerless deletion. This allows the marker bar to be used again in a new transformation round. DSBs, double-strand breaks; M, mutant

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