A CRISPR Cas12a/Cpf1 strategy to facilitate robust multiplex gene editing in Aspergillus Niger

Abstract

Background: CRISPR technologies have revolutionized strain engineering of Aspergillus species, and drastically increased the ease and speed at which genomic modifications can be performed. One of the advantages of CRISPR technologies is the possibility of rapid strain engineering using multiplex experiments. This can be achieved by using a set of different guiding RNA molecules (gRNA) to target multiple loci in the same experiment. Two major challenges in such experiments are firstly, the delivery of multiple guides simultaneously, and secondly, ensuring that each target locus is cut efficiently by the CRISPR nuclease. The CRISPR nuclease Cas12a, also known as Cpf1, presents a unique advantage to bypass this challenge. Specifically, and unlike Cas9, Cpf1 is able to release several gRNAs from a common precursor RNA molecule through its own RNase activity, eliminating the need for elements such as ribozymes or tRNA machinery for gRNA maturation. This feature sets the stage for much more straightforward construction of vectors for the delivery of many gRNAs, which in turn allows each locus to be targeted by multiple gRNAs to increase the odds of successfully inducing a break in the DNA.

Results: Here we present a toolbox that can be used to assemble plasmids containing a gRNA multiplex expression cassette, which is able to express a multi gRNA precursor. The precursor can be processed via Cpf1 RNase activity to produce multiple functional gRNAs in vivo. Using our setup, we have constructed plasmids that are able to deliver up to ten gRNAs. In addition, we show that three simultaneous deletions can be introduced robustly in Aspergillus niger by targeting each gene with several gRNAs, without prior gRNA validation or the use of genomically integrated selection markers.

Conclusion: In this study we have established an efficient system for the construction of CRISPR-Cpf1 vectors that are able to deliver a large number of gRNAs for multiplex genome editing in Aspergillus species. Our strategy allows multiple specific genomic modifications to be performed in a time frame of less than two weeks, and we envision this will be able to speed up cell factory construction efforts significantly.

Keywords: Aspergillus Niger; CRISPR gene editing; Cas12a; Cpf1; Fungi; Multiplexing.

Fig. 1

Strategy for Cpf1 multiplex CRISPR editing. ASchematic depiction of gene knock-out with a gRNA redundancy strategy. Multiple gRNAs are used to induce a double stranded break. Homology-directed repair using a dsDNA gene targeting substrate (GTS) as template induces a 300 bp deletion at the start of the gene.BSchematic for USER assembly of a Cpf1 CRISPR vector containing one gRNA using two USER compatible PCR fragments.CSchematic depiction of expression of a Cpf1 multi-guide precursor RNA from a MEC controlled by a U3 promoter and terminator, followed by processing of the precursor RNA by Cpf1, RNase Z and tRNase P to yield mature gRNAs.DSchematic USER assembly of a 9-gRNA Cpf1 MEC using two USER compatible PCR fragments and five gRNA bio-blocks. Graphics legend is presented in the box

Fig. 2

Three-target Cpf1 multiplex gene deletions inA. niger. ATransformations with three-target Cp1 multiplex vectors (pCpf1.MEC1, pCpf1.MEC2 or pCpf1.MEC3), without GTS molecules.BCo-transformations with three-target Cpf1 multiplex vectors and appropriate GTS molecules

Fig. 3

Rate of genotype frequencies found in triple deletion transformations based on diagnostic PCR. ‘Additional amplicons’ represents a wild-type or aberrant amplicon in addition to the deletion amplicon. No transformants without deletions were observed. (pCpf1.MEC1 n = 21, pCpf1.MEC2 n = 21, pCpf1.MEC3 n = 18)