Letter Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX
pubs.acs.org/synthbio
5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger Xiaomei Zheng,†,‡ Ping Zheng,*,†,‡ Kun Zhang,†,‡,§ Timothy C. Cairns,∥ Vera Meyer,∥ Jibin Sun,*,†,‡ and Yanhe Ma† †
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China § University of Chinese Academy of Sciences, Beijing, 100049, China ∥ Department Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, 13355, Germany ‡
S Supporting Information *
ABSTRACT: The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi. KEYWORDS: 5S rRNA, Aspergillus niger, CRISPR/Cas9 system, genome editing
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enable numerous genome editing options for the end user. Because of this high flexibility, simplicity, and efficiency, CRISPR/Cas9 systems have been rapidly adapted for genome editing and transcriptional regulation in various species.1,5−7 In eukaryotes, the sgRNA expression cassette requires careful design to ensure the formation of functional Cas9−sgRNA complex.8 Establishing a suitable promoter for sgRNA expression is a significant technical challenge when this system is first established in a certain organism, and often requires concomitant testing of multiple putative promoters which have high variations in gene editing efficiency.9 Various strategies
he Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) system has been demonstrated to be a powerful and revolutionary genome editing tool,1,2 as it holds a lot of promise to rationally modify the genomes of various species. In this system, the endonuclease Cas9 is guided to genomic target sites by complementary base pairing of an artificial single guide RNA (sgRNA). The sgRNA is composed of 20 bases of targeting sequence at the 5′-end, which is required next to an ∼3 nucleotide protospacer adjacent motif (PAM, commonly the base pairs -NGG).3 After targeting at the recipient genome by sgRNA, Cas9 mediates a double strand break (DSB). In eukaryotic cells, this DSB is usually repaired by either the nonhomologous end joining (NHEJ) or homologous recombination (HR) pathways.4 These two DNA repair mechanisms © XXXX American Chemical Society
Special Issue: Genome Engineering Received: December 17, 2017 Published: April 24, 2018 A
DOI: 10.1021/acssynbio.7b00456 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology
Figure 1. 5S rRNA based sgRNA expression cassette enables the CRISPR/Cas9 system to disrupt the albA gene in A. niger. (A) Schematic diagram of albA disruption mediated by NHEJ using CRISPR/Cas9 system based on U6 and 5S rRNA promoters. PhU6 promoter represented the promoter of human RNU6-1 gene (NR_004394); PanU6 promoter represented the 412-bp upstream of A. niger RNU6 gene (AY136823.1). 5S rRNA (−338) represented 5S rRNA gene contained its internal promoter along with its 338-bp upstream sequence. 5S rRNA represented 5S rRNA gene used as the internal promoter without upstream sequence. HDV represented the HDV ribozyme, and T6 represented a string of six thymines serving as RNA polymerase III terminator. Linear sgRNA constructs and Cas9 expression plasmid pCas9 were cotransformed into protoplasts of A. niger. Without any donor DNA, the DSBs induced by Cas9 were repaired by error-prone NHEJ system, which resulted in the albA disruption. (B) The prediction of different genetic elements of the 5S rRNA gene and its upstream sequences in A. niger, and the six-truncation setup. The predicted regulatory elements of this 5S rRNA gene contained TFIIIA binding element (A-box, +51 to +62) and TFIIIC binding element (C-box, +82 to +92), TATAlike box (−33 to −22), proximal sequence element (PSE, −65 to −46) and distal sequence element (DSE, −96 to −88). (C) albA gene disruption efficiency of the transformants with sgRNA constructs driven by different promoters. Bars represented the percentage of albino colonies that showed the albA disruption phenotype on the primary transformation plates (mean ± SD; n = 3).
virus ribozyme (HDV) or hammerhead ribozyme could yield a functional sgRNA after self-processing,17 but more cloning efforts and experimental workload are required. Finally, some tRNA promoters were also employed for driving sgRNA expression.18−21 However, gene editing efficiency of different tRNAs promoters varied widely in a strain-dependent manner.20 In some cases, the genomic editing efficiency based on tRNA is still disputed.18,22 In Yarrowia lipolytica, the use of tRNA as sgRNA promoter brought gene disruption efficiency only 30%, while fusion with other promoters, such as SCR1, resulted in much higher efficiency (92%).21 Rather than functioning as a promoter, tRNA is preferably used to excise the sgRNA from the primary transcript by its endogenous processing mechanism.21,23,24 Taken together, the choice of promoter for sgRNA expression represents a significant technical constraint for developing CRISPR/Cas9 gene editing systems. An important technical advancement would be a highly conserved, efficiently expressed promoter that lacks a mandatory transcriptional initiation nucleotide. Aspergillus niger is a well-established industrial cell factory able to produce organic acids and numerous industrial enzymes.25 Its extraordinary tolerance to extreme pH values (1.5−10) and its ability to hydrolyze many polymeric substances, make it an ideal cell factory for diverse biotechnological applications.25 Despite its industrial importance, high-throughput genetic tools are still missing, hampering the fundamental study and industrial improvement
have been used for initiating sgRNA transcription in eukaryotes. The promoter of U6 small nuclear RNA (snRNA) is among the most commonly used one.3 However, the U6 promoter requires a guanosine nucleotide to initiate transcription, thus limiting sgRNA spacer sequences to GN 19 NGG, and consequently reducing the available CRISPR/Cas target sites.10,11 Moreover, low sequence conservation of the U6 snRNA gene outside yeast and mammalian genomes has made it difficult to identify the U6 promoter in many species.12,13 Alternatively, heterogeneous U6 promoters can be used for sgRNA expression, but it often resulted in inefficient gene editing. For instance, Zheng et al.13 used the U6 promoter derived from Aspergillus oryzae to establish a CRISPR/Cas9 system in Nodulisporium sp, but the genome editing efficiency was only ∼16%. As an alternative, sgRNA was also prepared in vitro using T7 promoters,12 but sgRNA stability and uptake are significant limitations using this approach, which may influence efficiency.14 Moreover, since sgRNA gene is not genetically introduced into the cell, it is not suitable for situations that the presence of sgRNA gene, and its persistent or conditional expression are necessary, for example CRISPR-AID system mediated transcriptional activation, transcriptional interference, and gene deletion.15 Promoters recognized by RNA polymerase II have also been used to drive sgRNA expression. However, 5′ capping, and the long termination tail at the 3′ end, resulted in sgRNA that was highly inefficient in gene targeting (
