Gene repression via multiplex gRNA strategy in Y. lipolytica
- Jin-lai Zhang†1, 2,
- Yang-Zi Peng†1, 2,
- Duo Liu1, 2,
- Hong Liu1, 2,
- Ying-Xiu Cao1, 2Email authorView ORCID ID profile,
- Bing-Zhi Li1, 2,
- Chun Li1, 2 and
- Ying-Jin Yuan1, 2
© The Author(s) 2018
Received: 9 January 2018
Accepted: 13 April 2018
Published: 20 April 2018
The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest.
In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized.
Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.
Effective metabolic engineering of cell factories and technologies of genetics enables the production of biofuels and biochemical from renewable resources at low and competitive cost [1–7]. In this context, the oleaginous yeast Yarrowia lipolytica has become a very attractive cell factory for industrial biotechnology applications [8–19], because of its ability to natively accumulate high quantities of lipids coupled with a wide substrates portfolios [20, 21] and simple industrial scale-up operations . In addition, Y. lipolytica is also recognized as a “generally regarded as safe” (GRAS) organism , which makes it a promising candidate platform for the production of high-value pharmaceutical compounds [23–26]. In order to create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, a number of gene expression and deletion tools have been established in the last decade [27–33]. However, as a non-conventional yeast, the capability of selective and tunable perturbation of gene expression in Y. lipolytica is still rather undeveloped compared to other yeasts such as Saccharomyces cerevisiae, which hindered further complex design, engineering and application of this organism [34, 35].
The engineered CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system has been proved its ability of highly selective transcriptional modulation over a significant dynamic range [36, 37]. Two parts are involved in the CRISPR interference (CRISPRi) system: an endonuclease-deficient, yet RNA-binding Cas protein (dCas) and a single guide RNA (gRNA). The guide sequences in gRNA is responsible for specific recognition of target gene, but the target site was determined by a PAM (Protospacer Adjacent Motif) sequence, which varied from different Cas protein . Catalytically deactivated Cas9 of Streptococcus pyogenes (spdCas9) derived from a type II CRISPR system is the best studied and most widely used Cas protein [39–41]. The recent development of CRISPRi/dCas9 technology for Y. lipolytica now allows enhanced homologous recombination efficiency without labored genetic knockouts and promises to be a potent tool for other metabolic engineering . However, The SpCas9 requires a G-rich PAM sequence (5-NGG-3) which is not always available in all chromosomes, more particularly in AT-rich regions . Another CRISPR-Cas protein Cpf1 provides a potential solution. Cpf1 belongs to the class II type V-A CRISPR-Cas system [43–47] and recognizes a T-rich PAM at the 5′-end of the protospacer sequence . Cpf1 makes a staggered double-strand break resulting in five-nucleotide 5′-overhangs distal to the PAM site , whereas Cas9 creates blunt ends proximal to the PAM site . It is worth mentioning that the Cpf1 PAMs also vary with different sources, which describe as 5′-TTTN-3′ (or 5′-TTTV-3′) for EeCpf1 , AsCpf1 [51, 52], LbCpf1  and 5′-TTN-3′ for FnCpf1 [45, 54]. These features propel Cpf1 as an attractive protein complementary to the Cas9 for genome editing and gene regulation [45, 54, 55]. Based on Cpf1 endonuclease mutants, CRISPRi/dCpf1 system was constructed and shown to mediate target gene repression effectively in both bacteria [50, 51] and plant cells .
When using CRISPRi technology to repress target genes, high-efficiency repression site was necessary. In either CRISPRi/dCas9 or CRISPRi/dCpf1 system, the repression efficiency was highly depended on the binding position of the gRNAs on the target gene. In bacteria and mammalian cell, this relationship was proved to follow certain rules. The dCas9 targeting non-template DNA strand of coding sequence with the nearest distance to transcription start site (TSS) demonstrated the most efficient gene repression in E. coli [36, 39, 56], Shewanella oneidensis MR-1 , cyanobacteria  and many other cells . The dCpf1 from Acidaminococcus sp. and Eubacterium eligens also showed site-dependent rules that stronger gene repression was achieved when it was targeted to the template strand rather than non-template strand in E. coli [50, 51]. But in yeast, the repression efficiency of different gRNA targeting was more complicated. Onge et al. found that gRNA efficacy depended on accessibility and location of the target region, and the best region of target gRNA was between the TSS and 200 bp upstream of the TSS in S. cerevisiae . Another CRISPRi study, however, yielded a different result that the scRNA targeting the TSS + 21 position of erg12 achieved the most efficient repression (~ 3-fold), different from the best region Onge et al.  found. And from testing a total of in silico designed 88 scRNAs on 12 native yeast promoters, Jensen et al. found that high-efficiency repression site was hard to determine .
Genes, strains and culture conditions
Strains used in this study were listed in Additional file 1: Tables S1. The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) , Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes , dCas9-KRAB , dCpf1-KRAB, sfGFP [60, 61], VioA, VioB, VioE , and vector assembled modules of pMCSCen1 , PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China). Escherichia coli Trans1-T1 was used for plasmid construction and propagation, which was cultured in Lysogeny broth (LB) medium at 37 °C at 250 rpm. Whenever required, 100 mg/l ampicillin or 50 mg/l kanamycin was added. The ATCC 201249 MATA stain of Y. lipolytica was cultured in either YPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete media (SC) (0.67% yeast nitrogen base, 0.2% amino acid mixture, and 2% glucose). All Y. lipolytica culturing was done at 28 °C, and a shaking speed of 250 rpm was used for all liquid cultures in 25 ml polypropylene tubes or 250 ml conical flasks.
Plasmid construction and transformation
Plasmids and primers used in this study are listed in Additional file 1: Tables S1 and Additional file 2: Table S2, respectively. All plasmids employed for gene expression in Y. lipolytica were centromeric replicative vectors based on plasmid pSl16-Cen1-1, which was initially modified to include a new multicloning site and redubbed pMCSCen1 . The fragments of dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB were inserted into vector pMCSCen1 via restriction enzyme digestion and ligation to form corresponding plasmids (PMCS-dCpf1, PMCS-dCas9, PMCS-dCpf1-KRAB, PMCS-dCas9-KRAB).
For single repression, the synthesized gRNA was incorporated into the PMCS-dCpf1, PMCS-dCas9, PMCS-dCpf1-KRAB, PMCS-dCas9-KRAB plasmids respectively (unique BbsI restriction site) via one-step Golden Gate assembly. All plasmids had been inserted into the gRNA expression cassette, which helped realize rapid plasmid construction to target any genomic locus of interest. Detailed plasmid assembly methods were shown in Additional file 3: Data S1.
For multiple repression, the synthesized gRNA was incorporated into the JLPC/N–n or JLRC/N–n plasmids first. The resulting vectors were digested with BsaI or BsaI and NotI together to allow for synthesis as separate gBlock. The dCpf1-Multi vector or dCas9-Multi was digested with BsaI, and the resulting digestion product was mixed with the separate gBlock above in a Golden-brick Assembly reaction to yield the final dCpf1-Multi-gRNA or dCas9-Multi-gRNA plasmid.
All enzymes and enzyme substrates were purchased from New England Biolabs. Plasmid constructions were performed in Escherichia coli Trans1-T1. Frozen-EZ kit was used for Y. lipolytica transformations and transformants of Y. lipolytica were selected and screened for on Sc-Ura or YPD-Hph (1% yeast extract, 2% peptone, 2% glucose, 0.04% hygromycin) or Sc-Ura-Hph (0.67% yeast nitrogen base, 0.2% amino acid mixture, 2% glucose and 0.04% hygromycin) agar plates.
All Y. lipolytica strains were activated in Sc-Ura medium for 24 h, then 500 μl of each culture suspensions were transferred in 25 ml polypropylene tubes containing 5 ml fresh Sc-Ura medium for 24 h. Finally, 1 ml of each culture suspensions were transferred in 250 ml conical flask containing 50 ml fresh Sc-Ura medium. The strains were grown at 250 rpm for 24 h at 28 °C. 1 ml suspensions of each conical flasks were centrifuged at 5000 rpm for 2 min to remove the supernatant and the cells were washed and resuspended with water. Fluorescence intensity (excitation: 488 nm and emission: 530 nm) was measured using a 96-well polystyrene plates (black plate, clear bottom) (Corning Incorporated 3603, USA) after dilution into the linear range of the detector by a multi-mode microplate reader (SpectraMax M2, Molecular Devices, USA) and cell density (OD600) was measured using cuvette by UV Spectrophotometer (TU-1810) respectively.
All Y. lipolytica strains were activated in Sc-Ura medium for 24 h, then 500 μl of each culture suspensions were transferred in 25 ml polypropylene tubes containing 5 ml fresh Sc-Ura medium for 24 h. Finally, 1 ml of each culture suspensions were transferred in 250 ml conical flask containing 50 ml fresh Sc-Ura medium. The strains were grown at 250 rpm for 48 h at 28 °C. 3 ml suspensions of each conical flasks were centrifuged at 13,300 rpm for 10 min to remove the supernatant and the cells were washed and resuspended with 600 μl absolute ethanol. After adding glass beads (SIGMA), the mixture was shaked for 20 min, then was centrifuged at 13,300 rpm for 15 min to acquire the supernatant. Pigment intensity (absorbance: 584 nm) was measured using a 96-well polystyrene plates (black plate, clear bottom) (Corning Incorporated 3603, USA) by a multi-mode microplate reader (SpectraMax M2, Molecular Devices, USA) and cell density (OD600) was measured using cuvette by UV Spectrophotometer (TU-1810) respectively. The relationship between PVA content and relative absorbance was shown in Additional file 4: Fig. S1.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated from the mid-log phase cultures using the Bacterium Total RNA Extraction Kit (APEXBIO, China), according to the instruction of the manufacturer. And cDNA was synthesized using the GoScrip™ Reverse Transcription System (Promega, USA). Quantitative analyses of expression of target genes were achieved by SsoAdvanced™ SYBR® Green Supermix (Bio-Rad, USA). Gene act was used for normalization. Samples were tested in triplicate using the listed primers (Additional file 5: Table S3). The data were analyzed using the 2−ΔΔCT method.
Results and discussion
CRISPRi mediated gene repression based on four repressors in Y. lipolytica
To establish an effective reporter system in Y. lipolytica, several available fluorescent proteins were evaluated, including RedStar2, YFP  and GFP [60, 61]. The TEF intron (TEFin) promoter was applied to express these fluorescence genes, and the reporter functionality was determined by a multi-mode microplate reader. Of these variants, only GFP imparted remarkable fluorescence (Additional file 6: Fig. S2). Then the gfp reporter gene was integrated into rDNA locus forming various control strains (Additional file 7: Data S2). Six strains were selected randomly and measured by the multi-mode microplate reader as shown in Additional file 6: Fig. S2. The best performed strain (GFP-6) was selected and used as control strain called YL-GFP for expressing CRISPRi plasmids in the following works.
Enhanced gfp repression by one-step Golden-brick assembly of multiplex gRNAs
When using CRISPRi technology to repress target genes, specific binding sites were necessary for high-efficiency repression. Qi et al.  showed that a dCas9 with D10A-H841A mutations arrived 300-fold repression for gene silencing when targeting the non-template DNA strand in E. coli, and Gilbert et al. achieved 53-fold repression efficiency with addition of Mxi1 repressor in eukaryotic cells . However, as mentioned above, the repression efficiency in Y. lipolytica only arrived 3.1-fold with dCpf1-gRNA complex and 4.5-fold with dCas9-gRNA complex, which was pretty low compared with other strains. Furthermore, it seemed impossible to achieve great repression efficiency only by a simple target position in Y. lipolytica based on results above, but extra target sites screening often mandated a significant investment of time and effort. Therefore, developing a convenient multiplex targeting system in Y. lipolytica would be useful.
Selective and tunable perturbation of gene expression is a fundamental enabling technology in the fields of systems biology and synthetic biology, allowing the design of intricate synthetic circuits and the interrogation of complex natural biological systems [65–67]. Here, we described a multiplex CRISPRi system for multiple gene repression by one-step Golden-brick assembly in the oleochemical-producing yeast Y. lipolytica. The Golden-brick assembly method developed in this study provided a simple and more convenient way for plasmid construction than other tools. Biobrick standard assembly requires a step-by-step process using restriction sites and needs four restriction enzyme sites, whereas Golden-brick assembly in this study only needs two restriction enzyme site and all parts can be assembled in one step . Compared with assembly methods based on homologous recombination like Gibson, the Golden-brick assembly method can assemble different parts without the procedure of PCR in advance, which avoids introducing new errors in the process of PCR amplification . In conclusion, multiplex CRISPRi system provided the benefit of improving the regulation efficiency and gave a better strategy to rapidly inhibit target gene expression without the need of screening a large number of target sites in advance in Y. lipolytica.
Multiplex gene interference in Y. lipolytica
In metabolic engineering, balancing the expression level of multiple genes is crucial for increasing the productivity of biosynthetic pathways and subsequently for sustainable production of valuable products [57, 67]. As an attractive candidate for industrial biotechnology applications, Y. lipolytica has been widely used for production of oleochemicals [8, 14, 70, 71], biofuels [8, 16, 72, 73] and acetyl CoA-derived metabolites [9, 10, 11, 23, 74]. But the library of available tools is not as developed as that of other yeasts such as Saccharomyces cerevisiae [34, 35, 54], especially for multiplex gene repression simultaneously [59, 75].
In most cases, for multiplex gene regulation or epigenetic modifications, multiple gRNAs may need to be independently expressed, and the construction procedure is time-consuming . By using a multiplexed gRNA targeting strategy, we achieved efficient transcriptional simultaneous repression of several targeted genes in one step. In this study, we have demonstrated that the multiplex CRISPRi system could be used for PVA and GFP regulation solely or simultaneously, which promised to be a potent transformative tool that will be extremely valuable for metabolic engineering requiring throttled flux through essential pathways in Y. lipolytica.
In this work, we demonstrated successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica. By using a multiplexed gRNA targeting strategy, efficient transcriptional simultaneous repression of several targeted genes and different sites of one gene was achieved in one step without the need of screening a large number of target sites. This study thus paves a new avenue to facilitate metabolic engineering, synthetic biology and functional genomic studies of Y. lipolytica.
JLZ, YZP performed the experiments. YJY, YXC, BZL, CL, DL, HL, JLZ and YZP conceived the project and wrote the manuscript. All authors read and approved the final manuscript.
The authors are grateful for the financial support from the Ministry of Science and Technology of China (“973″Program: 2014CB745100), the National Natural Science Foundation of China (Major Program: 21621004; Foundation for Young Scholars: 21506153).
The authors declare that they have no competing interests.
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Data will be made available from the corresponding author on reasonable request.
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The Ministry of Science and Technology of China (“973″Program: 2014CB745100).
The National Natural Science Foundation of China (Major Program: 21621004; Foundation for Young Scholars: 21506153).
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