CRISPR/Transposon gene integration (CRITGI) can manage gene expression in a retrotransposon-dependent manner

The fine-tuning of gene expression contributes to both basic science and applications. Here, we develop a novel gene expression technology termed CRITGI (CRISPR/Transposon gene integration). CRITGI uses CRISPR/Cas9 to integrate multiple copies of the plasmid pTy1 into Ty1 loci, budding yeast retrotransposons. The pTy1 plasmid harbors a Ty1 consensus sequence for integration, a gene of interest with its own promoter and a selection marker gene. Interestingly, the expression of the pTy1 gene in Ty1 loci could be induced in synthetic complete amino acid depletion medium, which could activate the selection marker gene on pTy1. The induction or repression of the gene on pTy1 depended on Ty1 transcription. Activation of the selection marker gene on pTy1 triggered Ty1 transcription, which led to induction of the gene on pTy1. The gene on pTy1 was not transcribed with Ty1 mRNA; the transcription required its own promoter. Furthermore, the trimethylation of histone H3 on lysine 4, a landmark of transcriptionally active chromatin, accumulated at the 5′ end of the gene on pTy1 following selection marker gene activation. Thus, CRITGI is a unique gene regulation system to induce the genes on pTy1 in amino acid depletion medium and utilizes Ty1 transcription to create a chromatin environment favorable for the transcription of the genes on pTy1.

. CRISPR/Transposon gene integration (CRITGI) can introduce multiple plasmids into Ty1 loci. (a) A diagram of CRITGI. Two types of plasmids (pTy1 and PHM663) were simultaneously transformed into yeast. Blue colored bases and red colored bases indicate the target sequence and 5′ PAM sequence of gTy1, respectively. Marker: marker gene for selection of transformants. (b) Percentage of correct integration of the pTy1 plasmid into Ty1 loci. YIplac128 was used as a vector. (c) Comparison of the integrated pTy1 plasmid number in Ty1 loci between the linear plasmid and CRITGI. The Sal I-digested linear pTy1 plasmid (linear DNA) and the mixture with the pTY1 and PHM663 plasmids (CRITGI) were transformed into yeast cells. The number of integrated pTY1 plasmids on Ty1 loci was calculated for the ACT1 gene as one copy gene per genome by using real time (RT)-PCR, which included transformants derived from linear DNA (n = 8) and those from CRITGI (n = 20).
introduce multiple copies of pTy1 plasmids into Ty1 loci (Fig. 1c: CRITGI). Thus, CRITGI enables multiple copy numbers of pTy1 plasmids to integrate into Ty1 loci, as in Di-CRISPR 12 . Furthermore, we examined whether CRITGI simultaneously enabled several different types of pTy1 plasmids (3 species) to integrate into Ty1 loci. We obtained transformants with several species of pTy1 plasmids, in which the frequency of plasmid combination was reduced to 50% (2 pair) and 10% (3 pair) among the total transformants examined (n = 10) (Fig. S4D). Thus, CRITGI is able to introduce not only multiple copies of pTy1 plasmids but also various species of pTy1 plasmids into Ty1 loci.
Several studies have claimed that the gene inserted in the Ty1 element may undergo transcriptional cosuppression, which is defined as high gene copy number-triggered homology-dependent gene silencing 22,23 . Therefore, we examined whether a gene in the pTy1 plasmid within Ty1 loci was able to express or not. The pTy1-H3 plasmid harbors the HHT1 gene encoding the histone H3 protein with a FLAG epitope tag at the N-terminus driven by the strong constitutive TDH3 promoter (Fig. 2a: top) 24 . An immunoblot using an α-FLAG antibody showed that either a faint or undetectable level of FLAG-H3 was expressed in cells with various copies of pTy1-H3 plasmid grown in yeast extract-peptone-dextrose (YPD) medium, a conventional rich medium (Fig. S5A,B). These results suggest that FLAG-H3 expression is downregulated within the Ty1 element. The pTy1-H3 plasmid harbors the HIS3 gene as a marker gene, which is activated in cells grown in synthetic complete histidine dropout medium (SC-His). We hypothesized that activation of the HIS3 gene might induce Flag-HHT1 gene transcription from the pTy1-H3 plasmid. To test this hypothesis, we examined the expression of the FLAG-HHT1 gene in cells with pTy1-H3 grown in SC-His medium. Surprisingly, FLAG-H3 was detected in cells with pTy1-H3 grown in SC-His but not in YPD (Fig. 2a (CRITGI); lanes 1 and 2), although FLAG-H3 expression was detected in both SC-Ura (uracil dropout) and YPD using cells with the FLAG-HHT1 gene and the TDH3 promoter integrated in the URA3 locus ( Fig. 2a (URA3); lanes 3 and 4). Thus, marker gene activation triggers the expression of the gene in cells with the pTy1 plasmid, and this transcriptional repression correlates with the Ty1 element. FLAG-H3 expression in cells with pTy1-H3 was induced in SC-His only, but not in SC-Met (methionine dropout) or SC (complete set of amino acids essential for budding yeast) (Fig. S6). This means that the gene expression accompanying marker activation is severely restricted to the kind of amino acid. We next examined whether the protein expression level was proportional to the integration copy number of the pTy1 plasmid. The pTy1-V plasmid can express Venus, a variant yellow fluorescent protein (YFP), with a FLAG epitope tag at the N-terminus under a synthetic promoter (Psyn) and synthetic terminator (Tguo1) (Fig. 2b: top) [25][26][27] . In cells with the pTy1-V plasmid grown in SC-Leu (leucine dropout), FLAG-Venus was rarely detected in cells with a single copy of the pTy1-V plasmid (Fig. 2b bottom: lane 1). In cells with 3 copies of the plasmid, a faint band of FLAG-Venus was detected, and the signal intensity of the Flag-Venus band reached a plateau in cells with both 6 and 13 copies of the pTy1-V plasmid, although FLAG-Venus was rarely detected in cells with 13 copies of pTy1-V plasmids grown in YPD ( Fig. 2b bottom: lane 2 to 5). Thus, protein expression levels are proportional to the copy number of pTy1 plasmids. Next, we examined whether the gene transcription and protein expression from the plasmid were linked to Ty1 transcription. Cells with pTy1-V plasmids (10 copies) were cultured in YPD and then released into SC-Leu. Real-time quantitative PCR (RT-qPCR) was used to monitor the mRNA levels of Ty1, FLAG-Venus, and LEU2 (ACT1 as a reference). Ty1, LEU2 and Venus mRNA levels simultaneously increased after cells were released into SC-Leu ( Fig. S7A-C). Similar to the fluctuation of Venus mRNA, an immunoblot using an α-FLAG antibody detected the FLAG-Venus protein from 2 h after the cells were released into SC-Leu ( Fig. 2c: lane 1 to 4). Thus, FLAG-Venus expression correlated with an increase in Ty1 mRNA together with LEU2 gene induction. Next, cells with the pTy1-V plasmid were cultured in SC-Leu and then released into YPD. The mRNA levels of Ty1, LEU2 and Venus simultaneously decreased after being released into YPD medium ( Fig. S7D-F). The FLAG-Venus protein was also abolished during the time course, but its disappearance was delayed compared to FLAG-Venus mRNA ( Fig. 3d; lanes 1-4 and S7F), which is due to the robustness of the Venus protein 28 . Thus, the repression of the transcription of the Venus gene was also linked to the reduction in Ty1 mRNA, together with the inactivation of the LEU2 gene. Altogether, gene expression in the pTy1 plasmid is connected by Ty1 mRNA transcription.
Changes in Ty1 transcriptional factors might influence gene transcription in cells with the pTy1 plasmid. The SAGA complex, a general transcriptional activator, the Swi/Snf and ISWI chromatin remodeling complexes, and various other transcriptional regulators are involved in Ty1 transcription 20,[29][30][31][32][33][34] (Fig. 2f). Gcn5 is a general transcriptional activator, and the deletion of gcn5 reduces the transcriptional activities of both LTRs of the Ty1 elements and Psyn promoter 20,26 . The FLAG-Venus protein was rarely detected in the gcn5∆ background during the time course ( Fig. 2f; lane 4-6). Gcn4 also regulates Ty1 transcription and binds five potential Gcn4-binding motifs (5′-TGAATG-3′) in the vicinity of the LTR region of the Ty1 element (Fig. 2f). However, the Ty1 mRNA level in gcn4∆ cells remains at almost the same level as wild-type cells 34 . In contrast to the reduction in the FLAG-Venus protein in gcn5∆ cells ( Fig. 2f; lane 4-6), the FLAG-Venus protein was constantly expressed in gcn4∆ cells during the time course ( Fig. 2f; lane 1-3). These results suggest that Ty1 mRNA transcription controls gene expression in the pTy1 plasmid within the Ty1 element.
Jiang reported that transcription of the in-frame Ty1-gene fusion depends on the LTR in the Ty1 element 23 . The FLAG-HHT1 gene is in-frame with Ty1 transcription in cells with the pTy1-H3 plasmid. If FLAG-HHT1 mRNA occupied a part of the Ty1 mRNA, FLAG-HHT1 could be expressed without its own promoter (i.e., the TDH3 promoter in the pTy1-H3 plasmid). We tested Flag-H3 expression in both cells with the pTy1-H3 plasmid without the TDH3 promoter region (pTy1-H3∆p) and cells with the pTy1-H3 plasmid following the marker gene induction ( Fig. 3a: top). FLAG-H3 was rarely detected in cells with the pTy1-H3∆p plasmid regardless of the integration copy number of the pTy1-H3∆p plasmid, although FLAG-H3 was detected in cells with pTy1-H3 ( Fig. 3a: bottom). This suggests that the expression of FLAG-H3 in the pTy1-H3 plasmid needs its own promoter within the Ty1 element. From this result, we hypothesized that marker gene induction could "open" the Ty1 chromatin structure, which is favorable for gene transcription within the pTy1 plasmid. To demonstrate this hypothesis, we performed Micrococcus nuclease (MNase)-chromatin immunoprecipitation (ChIP) to examine the level of www.nature.com/scientificreports www.nature.com/scientificreports/ trimethylation of histone H3 on K4 (H3-K4me3) in chromatin surrounding the gene on the pTy1-V plasmid 35,36 . Histone H3-K4me3 is a landmark of active gene transcription and is enriched at the 5′ end of the active gene in budding yeast 35,36 . We compared the level of histone H3-K4me3 on the nucleosome both at the start position of Ty1 (Ty1 500) and 5′ end of the Flag-Venus gene (Flag-V (N)) on the pTy1-V plasmid ( Fig. 3b; upper model) in cells with pTy1-V plasmids (4 copies) cultured in SC-Leu and SC media. The level of histone H3-K4me3 in both Ty1 500 and Flag-V (N) regions was more significantly increased in the SC-Leu culture than in the SC culture, not in ACT1 region (Fig. 3b). This means that the repressed chromatin within Ty1 became more favorable for transcription following marker gene activation. If the chromatin structure within Ty1 was a detrimental factor for controlling gene transcription in the pTy1 plasmid, two different gene units set in tandem in the pTy1 plasmid could be synchronously expressed following marker gene induction. The pTy1-V-H3 plasmid harbors the (+P TDH3 and ∆P TDH3 , respectively) (top). Immunoblotting using an anti-Flag antibody detects Flag-H3 in cell extracts (bottom). Two types of HMY1496 strains (wild-type cells with the pTy1-H3 ∆p plasmid (I. P. No. = 2 and 28, respectively)) and the HMY1466 strain (wild-type cells with pTy1-H3 (I. P. No. = 14)) were cultured in SC-His at 25 °C overnight. As in Fig. 2, the nitrocellulose membrane, in which proteins had been transferred to, was stained with 0.1% Ponceau S buffer and scanned to quantify the protein level in each lane. After destaining the membrane by TBS-T, the same membrane was used for the Western blot using the α-Flag antibody. (b) MNase-ChIP assay using anti-histone H3-K4me3 antibody. The HMY1476 strain (wild-type cells with the pTy1-V plasmid (I. P. No. = 4)) was cultured either in SC or in SC-Leu (SC-L) at 25 °C for 5 h. Ty1 500 and Flag-V (N) were used as target sites for RT-qPCR analysis. *P < 0.05. Unpaired t-test (one-tail). Error bars represent the standard deviation of three biological replicates. The ACT1 gene locus was used as a control, and the H3-K4me3 level was not altered between SC and SC-L media. (c) CRITGI can synchronously express two types of genes set in tandem within Ty1 loci in SC-Leu, but not in YPD medium. A schematic representation of the pTy1-H3-V plasmid in the Ty1 element. The pTy1-H3-V plasmid harbors two gene sets: the FLAG-HHT1 gene with the Psyn promoter and Tguo1 terminator, FLAG-Venus gene with the Psyn promoter and Tguo1 terminator in tandem with the LEU2 marker gene (top). The HMY1500 strain (wild-type cells with the pTy1-H3-V plasmid (I. P. No. = 4)) was cultured either in SC-Leu (L) or YPD (Y) at 25 °C overnight. Immunoblots www.nature.com/scientificreports www.nature.com/scientificreports/ following arrayed genes in tandem: the FLAG-Venus gene and FLAG-HHT1 gene with the Psyn promoters and Tguo1 terminators (Fig. 3c: top). Both FLAG-Venus and FLAG-H3 were detected in cells with the pTy1-V-H3 plasmid grown in SC-Leu, although both proteins were rarely detected in YPD (Fig. 3c: bottom). The Flag-Venus level was lower than Flag-H3 (Fig. 3c: bottom) because Flag-H3 can be incorporated into chromatin after the replication fork passes 37 and the half-life of nucleosomal Flag-H3 extends longer than that of free Flag-Venus (Fig. 2d) 38 . Altogether, marker gene induction can alter the Ty1 chromatin environment, making it more favorable for gene transcription from the pTy1 plasmid.
We tested whether CRITGI could be applicable for metabolic engineering. Pyruvate decarboxylase (Pdc) plays an essential role in producing ethanol from pyruvate (Fig. 4a). Additional Pdc expression can increase ethanol production 39 . The pTy1-Pd plasmid possesses the PDC1 gene, a major gene among the PDC gene family, with the Psyn promoter and Tguo1 terminator and the LEU2 marker gene. Cells with the pTy1-Pd plasmid were cultured in SC-Leu (−L) and SC (+L) for 24 h and with wild-type (BY4742) cells in SC. The amount of ethanol released into the medium by cells with the pTy1-Pd plasmid (−L) increased more than wild-type cells, although the ethanol level in cells with the pTy1-Pd plasmid (+L) was almost similar to that in wild-type cells (Fig. 4b). These data demonstrate that the CRITGI system can produce the metabolite of interest by using amino acids.
In this research, we have developed a novel gene expression system named CRITGI. CRITGI can control gene expression in the pTy1 plasmid integrated into Ty1 by using marker gene activation. For maintaining plasmid in budding yeast, we usually use genes involved in amino acid syntheses and nucleotide synthesis as plasmid markers 18 . Together with conventional molecules that drive inducible promoters, for example, galactose for the GAL1/10 promoter 40 , cupper for the CUP1 promoter 41 and tetracycline for the tet promoter 42 , various amino acids or nucleotides are available to control gene expression by CRITGI, as long as the genes involved in their syntheses are applicable for the pTy1 plasmid marker genes. CRITGI uses CRISPR/Cas9 to integrate multiple copies of a plasmid into Ty1 loci. The integration number of a plasmid is proportional to the protein expression level. These advantages of CRITGI contribute to metabolic engineering. Fine-tuning the timing and levels of the expression of enzymes involved in both natural and engineered pathways can relieve bottlenecks and minimize the www.nature.com/scientificreports www.nature.com/scientificreports/ metabolic burden of chemical production 39,43,44 . The production of an end product requires the complete set of metabolic pathways of interest. The incomplete set of the metabolic pathway of interest, which lacks any enzyme steps, might cause the abnormal accumulation of metabolic intermediate(s) harmful for cell survival. CRITGI can induce multiple enzyme-encoding genes that are composed of the metabolic pathway and control the expression levels of enzymes. Although further development of the CRITGI system is needed in the future, CRITGI, which can express a set of enzymes of a metabolic pathway of interest, will contribute to industries such as metabolic engineering, materials engineering, and pharmaceuticals.
Native promoters in Saccharomyces cerevisiae regulated by carbon sources 45,46 or the availability of specific nutrients 47,48 are applicable to express or repress genes of interest in a trans-acting factor-dependent manner. CRITGI has a unique gene induction system; marker gene activation evokes Ty1 transcription, which is necessary to induce a gene on pTY1plasmid. We favor that Ty1 transcription alters the chromatin structure favorable for transcription of a gene from the pTy1 plasmid. (1) Genes on the pTy1 plasmid need their own promoters. (2) MNase-ChIP shows that H3-K4me3 accumulates at 5′ end of a gene in pTy1 following marker gene activation.
(3) Two genes that are set in tandem on the pTy1 plasmid can be synchronously expressed. Swi/Snf and ISWI chromatin remodeling complexes are involved in Ty1 mRNA transcription from the LTR together with transcriptional factors, such as Gcn5 20,33 , suggesting that chromatin remodeling factors may create a chromatin structure favorable for gene transcription from the pTy1-gene plasmid. CRITGI is a unique gene regulation system that alters the Ty1 chromatin structure from repressed to competent allowing gene transcription. Further efforts need to reveal the mechanism to create a chromatin environment favorable for gene transcription within the Ty1 element in the future.

Methods
Strains and media. The genotypes of the strains, plasmids and primers used in this study are listed in Table S1. The parental budding yeast strain used in the present study was BY4742 (MATα his3Δ leu2Δ1 met15Δ0 ura3Δ0) 49 . A yeast strain harboring a single gene deletion was commercially available from the haploid yeast open reading frame deletion collection 50 (GE Dharmacon, Lafayette, CO, USA). Yeast cells were routinely grown at 30 °C in YPD (1% yeast extract, 2% peptone, 2% glucose) or appropriate synthetic complete (SC) medium 51,52 . If necessary, the media were solidified by including 2% agar. For the time course analysis, yeast cells were cultured at 25 °C. A standard method was used for isolation of the yeast genome DNA 52 . E. coli strain DH5α 53 , and standard media and methods were used for plasmid manipulations 54 . For the isolation of plasmid free of dam methylation, INV110 E. coli competent cells (Thermo Fisher Scientific, Waltham, MA, USA) were used.

Plasmid construction.
To construct plasmid PHM661 (pTy1), the ~ 870 bp PCR product (Ty1 homologous recombination (HR) sequence, ranging from 1681 bp to 2541 bp in YPRWTy1-3) obtained using BY4742 genomic DNA as a template, with forward primer (HMP1196) and reverse primer (HMP1197), was digested with Bam HI and Hind III and ligated into Bam HI/Hind III-digested YIplac128 plasmid 55 . For the construction of plasmid PHM688, the Bam HI/Hind III-digested Ty1 PCR fragment was ligated into the Bam HI/Hind III-digested pRS403 plasmid 56 .
To construct the plasmid encoding Cas9 and crRNA, we used pML104 plasmid (gift from John Wyrick (Addgene plasmid # 67638)) as a base plasmid 57 . This plasmid requires the insertion of the 20-nt guide sequence within a single guide RNA (sgRNA) cassette. The construction of the sgRNA cassette in the pML104 plasmid was described elsewhere 6,57,58 . The target sequence was set within the Ty1 HR sequence for the guide RNA (gTy1 #3) (5′-ACGTCTTAGAACGGTCTGACGG-3′ (underline: PAM sequence)) and set within the short Ty1 HR sequence for gTy1 #4 (5′-ACCTACATACTGACATATTTGG-3′). To construct the 20-bp guide sequence within the sgRNA, two DNA primers (HMP1192 and HMP1193 for gTy1 #3; HMP1194 and HMP1195 for gTy1 #4) were mixed at a final concentration of 10 μM in annealing buffer (40 mM Tris-Cl pH 8.0, 20 mM MgCl 2 and 50 mM NaCl). The mixture was incubated at 95 °C for 5 min and cooled down for 90 min until it reached 25 °C. To obtain pML104 plasmid free of dam methylation because Dam methylation blocks Bcl I digestion, the pML104 plasmid was transformed into a dam-E. coli competent cell (INV110). To construct the PHM663 (gTy1 #3) and PHM664 (gTy1 #4) plasmids, 20-bp double strand DNA cassettes (gTy1 #3 and gTY1 #4) were ligated into Bcl I/Swa I-digested pML104 plasmid. The ligation mixture (2.5 µl of 25 ng/µl pML104, 0.5 µl of the annealed DNA strand, 0.1 µl of 10 units/µl Bcl I, 0.1 µl of 10 units/μl Swa I, 0.1 µl of Quick ligase, 1x T4 DNA ligase buffer (New England Biolabs, Ipswich, MA)) was run for 3 cycles of 5 min at 37 °C, 10 min at 16 °C. Two µl of the ligation mixture was transformed into DH5α E. coli competent cells to obtain the plasmids (PHM663 and PHM664). Construction of other plasmids is described in Supplementary method.
Yeast transformation for CRITGI. Yeast transformation was described elsewhere 59,60 . Yeast cells were grown on YPD solid plates, collected with toothpicks and suspended in 100 µl of one-step buffer containing a plasmid mixture 59 . The mixture with 0.5~1.0 µg of integration plasmid (LEU2 or HIS3 marker gene) and 0.5~1.0 µg of Cas9/gRNA plasmid (URA3 marker) was used for transformation. The cell suspension was incubated at 42 °C for 1 h and then plated on SC uracil and either leucine or histidine dropout medium (SC-Ura-Leu or SC-Ura-His). The transformants, plasmids, and parent strains are listed in Table S1. We routinely used the HMY1448 and HMY1459 strains, which have multiple copies of the PHM661 (LEU2) and PHM668 (HIS3) plasmids already integrated in Ty1 loci, respectively. These strains are useful in obtaining transformants harboring multiple copies of plasmids by transformation.
The isolation of chromosomal DNA from yeast transformants was described elsewhere 52 . To measure the number of plasmids integrated into Ty1 loci, we used the LightCycler 480 SYBR Green I Master (Roche Life Science, Penzberg, Germany). The mix contained 10 µl of 2x LightCycler 480 SYBR Green I Master Mix, 2 µl of 5 µM PCR forward primer, 2 µl of 5 µM PCR reverse primer and ~10 ng of total DNA (final volume 20 µl).