Cpf1 enables fast and efficient genome editing in Aspergilli

Katherina Garcia Vanegas,Zofia Dorota Jarczynska,Uffe Hasbro Mortensen,Tomas Strucko

doi:10.1186/s40694-019-0069-6

Katherina Garcia Vanegas, Zofia Dorota Jarczynska + Show 2 more

Open Access

https://doi.org/10.1186/s40694-019-0069-6

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Abstract

BackgroundCRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Moreover, the efficiency of the system often allows genetic engineering to be introduced in non-model species. The efficiency of CRISPR gene editing is due to the formation of specific DNA double-strand breaks made by RNA guided nucleases. In filamentous fungi, only Cas9 has so far been used as the CRISPR nuclease. Since, gene editing with Cas9 is limited by its 5′-NGG-3′ protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences in order to be able to target a larger repertoire of genomic sites. Cpf1 from Lachnospiraceae bacterium employs a PAM sequence composed of 5′-TTTN-3′ and therefore serves as an attractive option towards this goal.ResultsIn this study we showed that Lb_cpf1 codon optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. We have developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and in A. niger. Specifically, we used our setup to demonstrate that Cpf1 is able to catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting.ConclusionsIn this paper we introduce Cpf1 as a new tool in the fungal CRISPR toolbox. Our experiments demonstrate that Cpf1 can be efficiently used in Aspergilli for gene editing thereby expanding the range of genomic DNA sequences that can be targeted by CRISPR technologies.

Highlights

clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed
A restriction of CRISPR nucleases is the protospacer adjacent motif (PAM) sequence at the target site as the protospacer can be changed. This is important for CRISPR techniques that depend on accurate positioning of the DNA doublestrand break (DSB) for genetic engineering, e.g. for oligonucleotide directed mutagenesis or for tagging
We highlight that the PAM sequence of Cpf1 is 5′-TTTN-3′ [23] and it expands the repertoire of protospacers that can be used for CRISPR-mediated gene editing (Fig. 1)

Summary

Introduction

CRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Gene editing with Cas is limited by its 5′-NGG-3′ protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences in order to be able to target a larger repertoire of genomic sites. A restriction of CRISPR nucleases is the PAM sequence at the target site as the protospacer can be changed This is important for CRISPR techniques that depend on accurate positioning of the DNA doublestrand break (DSB) for genetic engineering, e.g. for oligonucleotide directed mutagenesis or for tagging. Vanegas et al Fungal Biol Biotechnol (2020) 6:6 Ca s9 PAM NGG vs Cp f 1 5' 3' PAM Another class 2 CRISPR nuclease Cpf ( known as Cas12a [22]) from Lachnospiraceae bacterium has recently appeared as a promising addition to the CRISPR toolbox [23, 24]. We present a vector-based Cpf platform for fungal CRISPR-mediated gene editing and demonstrate for the first time that Cpf can be efficiently used for gene editing in filamentous fungi

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Conclusion