CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

Yuan, Guoliang; Deng, Shuang; Czajka, Jeffrey J.; Dai, Ziyu; Hofstad, Beth A.; Kim, Joonhoon; Pomraning, Kyle R.

doi:10.3389/fbioe.2024.1452496

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

Journal Article · Wed Oct 16 00:00:00 EDT 2024 · Frontiers in Bioengineering and Biotechnology

DOI:https://doi.org/10.3389/fbioe.2024.1452496· OSTI ID:2476681

Yuan, Guoliang ^[1]; Deng, Shuang ^[1]; Czajka, Jeffrey J. ^[1]; Dai, Ziyu ^[1]; Hofstad, Beth A. ^[1]; Kim, Joonhoon ^[1]; Pomraning, Kyle R. ^[1]

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2476681

Report Number(s):: PNNL-SA--199630

Journal Information:: Frontiers in Bioengineering and Biotechnology, Journal Name: Frontiers in Bioengineering and Biotechnology Vol. 12; ISSN 2296-4185

Publisher:: Frontiers Media S.A.Copyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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