Combinatorial sgRNA library pool for gene cluster inactivation in Aspergillus nidulans

For combinatorial library construction, the first step involved designing individual sgRNAs targeting the core genes of 46 BGCs to induce stop codon mutations via CBE. We synthesized the corresponding 46 sgRNA expression cassettes (Table S2) and combined them into a sub-library pool. Subsequently, we constructed a combinatorial sgRNA library utilizing a tandem-arrayed tRNA-sgRNA expression system, enabling the simultaneous editing of up to five loci in a single transformation. To achieve this, the sub-library was PCR-amplified and assembled into the pUC19 backbone as tRNA-sgRNA arrays using the Golden Gate assembly method. To assess the coverage and uniformity of the 46 protospacers, the tRNA-sgRNA expression cassettes were PCR amplified using the combinatorial library as template. The pooled proto-spacer expression cassette was subsequently sequenced using next-generation sequencing (NGS).