Abstract

Introduction

The filamentous fungus Aspergillus niger is an important cell factory used in industry for the production of enzymes and organic acids. Owing to its genetic tractability, A. niger is widely used for research in fungal physiology, biochemistry and biotechnology. The availability of genome sequence of this organism [1,2] has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism [3,4]. The utility of A. niger as an industrial cell factory and as a model organism for research can be further improved by the development of a highly efficiency genome-editing system. https://www.ncbi.nlm.nih.gov/pubmed/17259976

Multiple nuclease-based gene targeting methods have been developed to improve the efficiency and precision of generating genetic modifications. These include Zinc-finger nuclease (ZFN) [5], Transcription Activator-Like Effector Nuclease (TALEN) [6], I-SceI meganuclease [7], and Clustered Regularly Interspaced Short Palindromic Repeat / associated nuclease 9 (CRISPR/Cas9) [8]. With the ability of nucleases to make DNA double strand break (DSB) in the host organism, nuclease-based gene targeting methods have been successfully used for gene disruption, knock-in mutation as well as improving heterologous protein production by integrating foreign genes to defined genomic loci [5,9,10]. The type II CRISPR/Cas9 system from Streptococcus pyogenes has revolutionized genome editing by providing a simple, flexible and efficient strategy to precisely edit the genomes of mammals [11], plant [12], fly [13], fungi [14] and bacteria [15]. Two components are essential in type II CRISPR/Cas9 system: a functional Cas9 nuclease and a chimeric guide RNA (gRNA) consisted of two regions, a CRISPR RNA (crRNA) harboring 20-nucleotide target-recognizing sequence at the 5’-end and a trans-activating crRNA (tracrRNA) for Cas9 binding. The crRNA is user-defined to match the target genomic locus. It guides the gRNA to form a RNA/DNA hybrid at the target genomic locus and recruits the Cas9 nuclease to generate DNA DSB [16]. Consequently, the CRISPR/Cas9 approach supports the editing of numerous genomic loci.

An efficient promoter that can facilitate gRNA transcription in vivo is a bottleneck of adoption of CRISPR/Cas9 system in Aspergilli. So far, in the reported CRISPR/Cas9 platform for A. niger, RNA polymerase II (Pol II) promoter and terminator were employed to drive the transcription of the gRNA, which required the inclusion of two self-splicing ribozyme sequences flanking the gRNA sequence to free the mature gRNA transcript [14]. The endogenous U6 promoter, an RNA polymerase III (Pol III) promoter, has been used for CRISPR/Cas9 genome editing in A. oryzae with an efficiency lower than 20% in generating gene mutations [17]. Another Pol III promoter, U3 promoter of A. fumigatus (AfU3), was successfully used to transcribe gRNA in A. nidulans. However, endogenous U6 and AfU6 promoters failed in generating functional gRNA in A. nidulans, which indicates that U6 promoters have limited function as gRNA promoters in fungi [18,19]. Therefore, it is important to find reliable promoters to drive gRNA transcription in vivo that can result in efficient genome editing of A. niger to expand its utility as a model and industrial organism.

In eukaryote cells, the transfer ribonucleic acid (tRNA) genes are transcribed by Pol III [20]. The transcription of fungal tRNA and other eukaryotes genes is constitutive and is independent of carbon source and cultivation conditions [21,22]. The tRNA promoter includes a region centered approximately 30 base pairs (bp) upstream of the tRNA gene start site and two intragenic sequences, Box A and Box B [20,23,24]. Their short sequences [25] and their self-splicing capacity [26,27] make tRNA promoters suitable for transcribing the short and non-coding gRNA sequence. The self-splicing ability of tRNA has been used to separate multiplexed gRNAs in plants [28] and recently in filamentous fungi [18]. To the best of our knowledge, using tRNA promoter to independently drive gRNA transcription has not been described in filamentous fungi. In this study, we describe the use of tRNA promoters and terminators to drive the gRNA transcription in vivo and show that this method is highly efficient in genome editing in A. niger.

Results

The use of tRNA promoters and terminators to drive gRNA transcription in A. niger

We manually curated the genome of the A. niger NRRL3 (http://gbrowse.fungalgenomics.ca/cgi-bin/gb2/gbrowse/Aspni_nrrl3_public) and identified 284 tRNA genes (chromosomal locations and sequences were listed in S1 Table). Six of the 284 tRNA genes are pseudogenes and three encode selenocystein tRNA. There are 8–24 tRNA genes for 17 of the 20 main amino acids. The three amino acids with fewer than eight corresponding tRNA genes are: tryptophan with one, asparagine with three, and cysteine with four tRNA genes (S1 Table). To determine the ability of tRNA promoters to drive the functional transcription of gRNA, we chose two random tRNA genes from every amino acid except that of asparagine and tryptophan. Three valine tRNAs were chosen due to the lack of intron in one and the difference in intron lengths in the two others. In total, 37 tRNA genes and their flanking sequences to use as promoters and terminators were selected (Table 1). Each gRNA cassette contains the following elements: 1) tRNA promoter which comprises the entire tRNA gene including introns, plus 100 nt upstream sequence; 2) the gRNA sequence that comprises a 20 nt crRNA guiding sequence that targets the albA gene (gene ID: NRRL3_00462) and a 80 nt tracrRNA sequence for Cas9 protein binding [29]; and 3) the 7–42 nt of tRNA terminator that spans from the end of tRNA gene to the poly-T stretch. The gene albA encodes a polyketide synthase involved in both dihydroxynaphthalene melanin and naphtho-gamma-pyrone synthesis that are required for spore pigmentation [30]. The disruption of albA prevents biosynthesis of black pigment, and results in white mutant conidia [14,30]. This phenotypic change provided a visual scoring of mutants directly on the transformation plate. For a quick, initial test of the ability of tRNA promoters and terminators to regulate gRNA transcription, we co-transformed the 37 linear gRNA cassettes independently into A. niger with cas9-bearing plasmid ANEp8-Cas9 (Fig 1A). The ANEp8-Cas9 plasmid was derived from the plasmid ANEp8 [31] that contains AMA1 sequence allowing extra-chromosomal replication [32] and the pyrG selection marker.

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Fig 1

The schematic representation of tRNA-driven CRISPR/Cas9 system adapted for A. niger.

(A) The ANEp8-Cas9 plasmid used to express cas9 gene. (B) The construct of gRNA cassette and the ANEp8-Cas9-gRNA plasmid bearing both gRNA cassette and cas9 gene. The tRNA promoter is composed of a tRNA gene and 100 bp upstream sequences. Arrow indicates the start site of transcription. Abbreviations: bla, beta-lactamase gene conferring ampicillin resistance; pkiA Prom, promoter of the pyruvate kinase gene; glaA Term, terminator of the glucoamylase gene; NLS: nuclear localization signal.

Table 1

Functional testing of 37 tRNA promoters and terminators in CRISPR/cas9 genome editing.

tRNA promoter	Length of tRNA gene (bp)	Length of intron (bp)	Anticodon	Length of tRNA promoter (bp)	Length of tRNA terminator (bp)	Number of total colonies in transformation plates*	Number of white colonies (albA mutant)
tRNAAla5	72	0	AGC	172	16	88	9
tRNAAla23	72	0	AGC	172	7	36	2
tRNAArg8	72	0	ACG	172	9	96	12
tRNAArg21	72	0	ACG	172	9	71	8
tRNAAsp2	91	19	GTC	191	18	124	12
tRNAAsp5	90	18	GTC	190	11	65	2
tRNACys1	72	0	GCA	172	11	100	7
tRNACys2	72	0	GCA	172	15	72	5
tRNAGln1	72	0	CTG	172	16	72	6
tRNAGln2	92	20	TTG	192	20	102	6
tRNAGlu1	72	0	TTC	172	17	56	4
tRNAGlu12	71	0	TTC	171	16	63	3
tRNAGly5	71	0	GCC	171	26	68	0
tRNAGly13	71	0	GCC	171	16	34	3
tRNAHis2	84	12	GTG	184	42	62	4
tRNAHis3	71	0	GTG	171	32	82	4
tRNAIle4	74	0	AAT	174	16	60	3
tRNAIle8	74	0	AAT	174	17	94	4
tRNALeu6	111	27	AAG	211	17	148	8
tRNALeu14	112	27	AAG	212	13	46	1
tRNALys6	73	0	CTT	173	16	124	6
tRNALys17	73	0	CTT	173	18	82	5
tRNAMet4	72	0	CAT	172	15	84	2
tRNAMet9	72	0	CAT	172	21	78	3
tRNAPhe2	73	0	GAA	173	17	60	2
tRNAPhe12	73	0	GAA	173	11	88	10
tRNAPro1	91	19	AGG	191	17	57	7
tRNAPro3	91	19	AGG	191	17	68	3
tRNASer4	105	23	GCT	205	13	98	4
tRNASer7	81	0	AGA	181	15	90	4
tRNAThr5	73	0	AGT	173	15	46	4
tRNAThr6	73	0	AGT	173	13	48	4
tRNATyr3	88	13	GTA	188	22	63	6
tRNATy7	88	13	GTA	188	21	42	2
tRNAVal1	74	0	CAC	174	18	33	4
tRNAVal4	96	23	CAC	196	25	45	1
tRNAVal15	87	14	TAC	187	18	64	7

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* The colonies number was from single transformations

In the control plate where the cells were transformed with ANEp8-Cas9 alone, only black colonies appeared (S1A Fig). Except in the case of tRNA^Gly5, all co-transformation yielded a mixture of black and white mutant colonies. These results suggest that almost all endogenous tRNA promoters and terminators are able to transcribe functional gRNA in the CRISPR/Cas9 system in A. niger. However, the co-transformation method resulted low inactivation rate of albA gene, ranging between 2 to 13% (Table 1). The relatively low rates of mutation using this co-transformation method is expected because the frequency of maintaining extra-chromosomal plasmid harboring the selectable marker is much higher than that of maintaining the integrated exogenous DNA [32]. In other words, only an uncontrolled subpopulation of transformants in these experiments is predicted to contain the linear gRNA cassette. Consequently, this initial experiment only reveals that most endogenous tRNA promoters and terminators are able to drive gRNA transcription and support CRISPR/Cas9 mutagenesis in A. niger, it does not provide quantitative information on the efficiency of different tRNA promoters in generating functional gRNA.

Discussion

Besides an active Cas9 protein, a functional gRNA is an essential component in CRISPR/Cas9 system to engineer the host genome. For delivering gRNA in vivo, the formation of functional gRNA requires an efficient promoter to drive gRNA transcription and a splicing mechanism to form mature gRNA transcript. In this study, we describe a strategy using Pol III tRNA promoters to drive gRNA transcription. This study shows that the self-splicing ability of tRNAs is highly efficient in generating functional gRNAs.

Since 2015, the CRISPR/Cas9 system in genome editing has been established in several genera of filamentous fungi. As summarized in Table 6, the promoter selection has shifted from Pol II promoter (e.g. pgdA and trpC promoters) to Pol III promoters for the in vivo transcription of gRNA. This is because Pol III promoter is more suitable for in vivo transcription of short non-coding transcripts such as gRNA [20,41]. In addition, the short length of Pol III promoters and terminators makes it easier to construct gRNA cassette. Among Pol III promoters used in filamentous fungi CRISPR/Cas9 platforms, the species-specific U6 promoter is the most widely used [17,42–47], and the SNR52 promoter from Saccharomyces cerevisiae has been tested in Neurospora crassa [48] and A. fumigatus [49] (Table 6). In this study, the tRNA promoters contain the entire sequence of tRNA gene plus 100 nt upstream sequence with a total sequence varying from 171 to 272 nt (S2 Table), which is similar as the lengths of U6 (200 to 250 nt) and S. cerevisiae SNR52 (269 nt) promoters [29,50]. The main challenge of current Pol III gRNA promoters such as U3 and U6 is that the same type of promoter results in highly variable gene editing efficiency amongst different species. Unlike U6 promoter that is an upstream element outside of the transcription start site [50], the tRNA gene is transcribed together with gRNA sequence [20,51]. In the cell the tRNA transcript is posttranscriptionally cleaved at the 3‘-end by RNase Z [27,28]. Accordingly, tRNA promoter regulated system is able to release mature functional gRNA transcript as demonstrated in this study and elsewhere [52].

By introducing upstream sequences, the tRNA promoters in our constructs include all external and internal binding sites of Pol III transcription factors needed for transcription to proceed normally. Except tRNA^Gln2, the tRNA promoters tested in this study lead to high rate of gene mutation (82–97%). In endogenous U6 promoter-driven platforms, the mutation rate is highly variable among different fungal species and ranges from 10 to 100% (Table 6). In a recent study, the endogenous U6 promoter and the heterologous AfU6 promoter failed to transcribe gRNA in CRISPR/Cas9 system in A. nidulans [18]. In addition, gRNAs transcribed by S. cerevisiae SNR52 promoter resulted in mutation rates of between 25 to 53% in A. fumigatus [49] (Table 6). These studies show that the type of promoters used to transcribe gRNA has a high impact in CRISPR/Cas9 activity. Overall results show that expressing Cas9 and the gRNA in single plasmid improve gene deletion and gene replacement. In addition no difference on growth was observed when transformed A. niger with ANEp8-Cas9 or ANEp8-Cas9-gRNA in the present study, except the color phenotype (S1A and S1B Fig). This study provides a broad list of highly efficient tRNA promoters for gRNA transcription.

In A. niger and other eukaryotes, DSB created in the genome is repaired by NHEJ or by homologous recombination [55]. The NHEJ is the most dominant pathway in A. niger and the frequency of homologous recombination increases with the deletion of the kusA gene, which is involved in the NHEJ repair mechanism [56]. In this study, we tested CRISPR/Cas9 mediated gene replacement by homologous recombination in both kusA^- and kusA⁺ strains. By targeting the glaA locus, we demonstrated that the CRISPR/Cas9 method can increase the frequency of gene replacement in A. niger (Table 5). In gene replacement mediated by CRISPR/Cas9 activity, our results are comparable to the results obtained in filamentous fungi such as A. nidulans, Trichoderma reesei and Myceliophthora thermophila, and show higher frequency comparing to findings in A. aculeatus, A. fumigatus, Penicillium chrysogenum and A. carbonarius (Table 6). In summary, the tRNA promoter-driven gRNA transcription system is very efficient to generate DNA double-strand break for gene mutation mediated by NHEJ and for gene replacement by homologous recombination in kusA⁺ and kusA^- strains. This powerful tool will facilitate further improvement of the industrial fungus A. niger.

Material and methods

Supporting information

Acknowledgments

Funding Statement

This work was supported by NSERC Strategic Industrial Biocatalysis Network. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

All gene sequences from A. niger and the genome sequence of A. niger NRRL3 are available at http://gbrowse.fungalgenomics.ca/cgi-bin/gb2/gbrowse/Aspni_nrrl3_public.

References