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How do transgenic transformations cause off-target phenotypes?

How do transgenic transformations cause off-target phenotypes?


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I read that transgene insertions can cause off-target mutations that result in a phenotype being overly ascribed to the transgene, e.g. the increase of lifespan being ascribed to Sir2 overexpression (Burnett et al, 2011, https://www.ncbi.nlm.nih.gov/pubmed/21938067). In that particular study, they outcrossed the transgenic strain several times with wild-type to remove these off-target effects, a mutation in Dyf2 being named in particular.

I have a poor background in genetics, so I don't really understand how transgene insertions can cause off-target mutations, nor how outcrossing can isolate the intended mutation.


The C.elegans used in the study you linked was originally made by Tissenbaum & Guarente using the gamma irradiation protocol.

In short, the protocol provides the injection of the desired DNA construct in the worm's gonad followed by gamma irradiation of the worms.

Upon radiation exposition, the genomic DNA of the worm will break at random sites and will undergo repairing by the appropriate cellular enzymes, during the latter stage the exogenous DNA can be integrated (ligated) into the genome. However, the genomic DNA can also acquire random mutations due to the radiation. This is how transgene insertions can cause off-target mutations in this case.

With other organisms or cells, the protocols are different. To engineer mammalian cells, for example, radiations are never used but still, you have to generate breaks in the genome to edit it. Depending on the method the "off-target rate" of a given experiment may vary, but it is never zero (not yet).


When you cross a mutant organism with a wildtype one (backcrossing), in each offspring half of the alleles originate from the mutant parent and half from the wildtype. Another way to look at this is to say that approximately half the offspring will contain any given mutation, assuming it is heterozygous in the mutant organism, and doesn't cause a fitness differential.

Because of this, on average, each time you backcross to the wildtype you lose half the mutations. The probability of a mutation remaining after several rounds of backcrossing reduces very quickly (0.5 after one generation, 0.25 after 2, 0.125 after 3, 0.0625 after 4, 0.03125 after 5 etc).

At each round of backcrossing you test offspring and select the ones that have the intended mutation and use these for the next round, so while the probability of keeping the intended mutation stays high (~1.0) the probability of keeping unintended mutations decreases very rapidly. This all assumes that the offtarget mutations are on different chromosomes, or far enough away from the intended mutation (unlinked) that they segregate separately and are not carried forward into the backcross generations by 'linkage drag'.


Genetic manipulation of Soc1-like genes promotes photosynthesis in flowers and leaves and enhances plant tolerance to high temperature

The rapid rise in mean global temperature as a result of global warming threatens plant productivity (Li et al., 2015 ). Chloroplasts and chloroplast proteins are associated with environmental stresses (Alexia et al., 2019 Hong et al., 2020 ). Many heat-shock proteins (HSPs) associate with chloroplast development and improve plant tolerance to heat stress at a high temperature (Shen et al., 2015 Zhong et al., 2013 ), whereas no gene is reported to promote chloroplast development and enhance tolerance to high temperature synchronously. The impact of high temperature on chloroplast is of particular significance since photosynthesis is often inhibited before other cell functions are impaired (Zhang et al., 2010 ). Thus, promoting chloroplast biogenesis and photosynthesis is a potential method to enhance heat tolerance of plants. We previously found that overexpression of SOC1 or SOC1-like genes in heat-stressed plants induces chloroplast biogenesis in petals (Wang et al., 2019 ). However, it is unknown whether the photosynthesis apparatus is impaired and whether the plant thermotolerance is enhanced in transgenic plants. In our present study, the transplastomic (harbouring GFP reporter gene driven by psbA promoter of chloroplast), multigene transgenic tobacco (Fbp21 gene was introduced to the genome of the pure line of GFP transplastomic tobacco-labelling nFbp21*pGFP) and transgenic petunia-overexpressing FBP21 gene were produced by chloroplast and nuclear transformation. Additionally, the transgenic plants (Fbp21-labelling F21 and Fbp21*22-labelling F21_22 in this paper) harbouring SOC1-like genes and RNA-Seq data of petals, previously reported (Wang et al., 2019 ), were also integrated. Finally, a series of experiments related to RNA sequencing in leaves, biological and physiological, anatomical and phenotypic determination were undertaken.

When plants were grown at high temperature (40°C days/28°C nights), it showed that only nonphotosynthetic plastids containing plastoglobules were seen in pink petals of control tobacco plants. We observed morphologically normal chloroplasts in green petals of the SOC1-like gene transgenic tobacco plants (Figure 1a). Chloroplasts in green petals of nFbp21*pGFP transplastomic tobacco were observed to emit red and green fluorescence simultaneously at high temperature (Figure 1b). It indicates that chloroplast genes were expressing in these heat stress-induced plastids in petals. Maximum photochemical efficiency values (Fv/Fm) determination also showed that photosynthesis took place in chloroplast-containing petals (Figure 1c and d). Most of the photosynthesis genes were dramatically up-regulated in chloroplast-containing green petals (Figure 1e). Immunoblot analysis also showed many photosynthesis associated proteins were synthesized in green petals (Figure 1f).

Heat-resistant assay showed that the SOC1-like gene transgenic tobacco (F21and F21_22) was substantially different from the control tobacco in their tolerance to prolonged extreme heat stress. For 2-week-old tobacco plants, more light yellow seedlings were seen in the wild-type tobacco than in transgenic lines after heat stress (Figure 1g). Fv/Fm values in transgenic lines were also notably higher than that of wild type (Figure 1g and h). The lower electrolyte leakage (EL) (Figure 1i) and higher survival rate (Figure 1j) suggest that these 2-week-old transgenic tobacco had enhanced heat tolerance. When plants were grown at normal temperature, more chloroplasts in cells of leaves of the transgenic tobacco (including F21 or F21_22 tobacco) also observed in the nFbp21*pGFP transplastomic tobacco according to red and green fluorescence compared to GFP transplastomic tobacco (Figure 1k). These results suggest that overexpression of Soc1-like genes promotes chloroplast biogenesis in transgenic leaves. When grown at high temperature, the leaf chloroplasts of the transgenic tobacco (also seen in nFbp21*pGFP) maintained normal appearance and orderly distribution and emitted more green fluorescence (Figure 1k). These observations indicate that the chloroplast genes can normally express at high temperature, whereas the chloroplasts in leaves of control tobacco became swollen, globular and irregular. It was consistent with what reported by Kwon and colleagues in GFP transplastomic tobacco (Kwon et al., 2013 ). The structural changes of chloroplasts and their scattered distribution in control tobacco (Figure 1k) suggest higher instability of varied cell membranes and cell damages by heat at high temperature.

The response to high temperature of 6-week-old tobacco plants was also markedly different between control and transgenic tobacco (Figure 1l). Many photosynthesis genes were dramatically up-regulated in leaves of transgenic plants growing at high temperature (Figure 1m). Immunoblot analysis showed that photosynthesis-associating proteins were accumulated in transgenic plants (Figure 1n). Under continuous heat stress (45°C for 9 h), Fv/Fm values were also significantly higher in leaves of transgenic tobacco plants (Figure 1o). A time series of net photosynthetic rate (Pn) determination indicated that the leaf Pn rate of transgenic tobacco plants was higher than that of wild type (Figure 1p). Magnesium is part of the chlorophyll and essential for photosynthesis (Leonard, 1954 ), and higher magnesium content was also detected in leaves of F21 transgenic tobacco plants (Figure 1q). Taking together, these results suggested that SOC1-like gene transgenic tobacco plants possess enhanced photosynthetic capacity under heat stress conditions.

Leaf EL value of 6-week-old transgenic tobacco was lower than that of wild type after heat stress (Figure 1r). RNA-seq analysis showed that genes encoding heat-shock proteins were also greatly up-regulated in leaves of transgenic tobacco plants under heat stress (Figure 1s). GC-MS analysis showed the proline content was significantly higher in SOC1-like gene transgenic tobacco plants (Figure 1t). It is worth noticing that during budding phase, the transgenic tobacco plants were not impaired and flowered normally under heat stress (12-h light cycle for 15 days). In contrast, wild-type tobacco plants did not flower or flowered poorly under the same heat stress condition (Figure 1u).

Additionally, the transgenic petunia-overexpressing Fbp21 gene was also more heat tolerant at varied growth phases than wild-type petunia plants (Figure 1v and w). The highest survival rate (96.60%) was recorded for 2-week-old transgenic petunia after heat stress (Figure 1v). For 6-week-old petunia seedlings, none of the wild-type petunia seedlings survived after 3 days under high temperature (Figure 1w). Similar to tobacco-overexpressing SOC1-like genes, the transgenic petunia-overexpressing Fbp21 gene also produced light green petals under heat stress (Figure 1x).

In an earlier study, we reported for the first time that SOC1-like genes promote chloroplast biogenesis in heat-stressed petals (Wang et al., 2019 ). In the present study, we show that cell containing increased number of chloroplasts is observed more frequently in leaves of Soc1-like gene transgenic plants than in wild-type plants and that SOC1-like genes up-regulate photosynthesis and heat-shock-associated genes, improve plant photosynthesis and alleviate heat stress damage to the chloroplast. Our results demonstrated that the super plants having chloroplast-containing petals, higher chlorophyll contents, increasing photosynthesis and enhancing heat tolerance could be synchronously achieved by genetic engineering. We showed that plant flowers can perform photosynthesis to further improve carbon utilization efficiency under heat stress and that overexpression of SOC1-like genes reduce the deleterious effects of heat stress on chloroplast and enhance photosynthesis in plants. Our observation provides a novel insight into the crosstalk mechanism between high temperature, plant functional chloroplast biogenesis, plant photosynthesis and plant heat tolerance. Producing heat-tolerant plants will be of great ecological and economic significance under the increasing threat of global warming.


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Results

The pNP system works efficiently in soma and germline

The conditional transgenic RNAi systems in Drosophila can temporally and spatially knock down target genes with the binary Gal4/UAS system. Without Gal4, the hairpin encoded by the DNA sequence downstream of the UAS would not be expressed, enabling us to generate and maintain thousands of transgenic RNAi lines based on the pVALIUM vectors 2,4 . However, we noticed that 15.8% of the transgenic RNAi lines based on the pVALIUM20 system in the Tsinghua Fly center (THFC) cannot maintain homozygosity (Supplementary Data 1). Meanwhile, 8.5% of the ɸC31 and long double-stranded RNA (dsRNA) hairpin based transgenic RNAi library (KK collection) in the Vienna Drosophila Resource Center (VDRC) are heterozygous, and 17.9% of the pVALIUM20 system based transgenic RNAi lines in the VDRC are heterozygous (Supplementary Data 1). All these vectors used in the previously developed transgenic RNAi systems are based on the pUAST vector, whose promoter consists of tandem UAS repeats followed by the TATA box from the Drosophila hsp70 gene 1,4 . Given that the hsp70 basal promoter can direct transcription without Gal4 induction in certain tissues at the standard 25℃ 9 , we speculated that the lethality of these homozygous transgenic RNAi lines could be due to the leaky activation of the hsp70 basal promoter.

To test this possibility, we replaced the hsp70 basal promoter in the pVALIUM20 vector with a Drosophila synthesized core promoter (DSCP) 13 . Since the hsp70 basal promoter is longer than DSCP, we added a 100 bp spacer sequence upstream of the UAS to ensure the appropriate working distance between the promoter and gypsy insulators. To achieve precise processing and expression of multiple shRNAs 14 , we amplified an intergenic linker between miR-2a-1 and miR-2b-2 and cloned it downstream of the miR1 scaffold. With these optimizations, we designated the resulting construct as the pNP vector (Supplementary Fig. 1).

We designed and generated stable transgenic lines using this system to express shRNAs targeting a set of protein-coding genes with known loss-of-function phenotypes in the wing, eye, neurons, intestine, testis and ovarian stem cell system. Firstly, knockdown of egfr, Hp1a, kis, Notch in the wing driven by wing specific Gal4s generated severe wing defect (Fig. 1a), which is consistent with previous description 4,15,16,17,18 . Furthermore, targeting of white, light, hedgehog (hh) using the ey-Gal4 produced the same eye phenotype as previous reports (Fig. 1b) 19,20,21 , suggesting the pNP transgenic RNAi system can efficiently deplete genes in soma. We also chose several genes with known loss-of-function phenotypes to analyze the efficiency of pNP system in the neurons. Consistent with previous reports 1,22,23 , all of them caused lethality when driven by elav-Gal4, indicating that pNP works efficiently in neurons (Supplementary Fig. 2a). Next, we tested the efficiency of the pNP system in intestine, testis and ovarian stem cell system. We used the germ cell specific nos-Gal4 to knock down piwi or bam, which severely reduced ovary size compared with the control (Fig. 1c), and resulted in flies that did not lay eggs (Fig. 1d). In addition, RNAi of bam increased the number of germline stem cells (GSCs) as previous reports 24 , while knocking down of punt in testis by nos-Gal4 showed no GSCs and less germ cells 25 , and depletion of Notch in intestine by esg-Gal4 increased the number of intestinal stem cells and enteroendocrine cells (Supplementary Fig. 2) 26 . Taken together, these results show that the pNP transgenic RNAi system can efficiently and specifically knock down target genes in not only soma, but also neurons, intestine, testis and ovarian stem cell system.

The pNP system works efficiently in soma and germline. a Examples of pNP-induced wing phenotypes using the MS1096-Gal4 or C96-Gal4. Scale bars, 500 μm. b Knock down of white, light or hh in the eye driven by ey-Gal4. Scale bars, 200 μm. c Dark field images of ovary phenotype, with knock down of piwi and bam controlled by nos-Gal4. Scale bars, 300 μm. d Fertility rates of control, pNP-piwi and pNP-bam female flies using the nos-Gal4 driver (n = 10, mean ± s.d.). Data are evaluated with one-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Significantly reduced leaky expression from basal promoter

To compare the basal activities of the promoters in the pNP and pVALIUM20 vectors, we replaced the miR1 scaffold with the luciferase gene as the reporter. First, we tested the leaky expression of luciferase in these transgenic flies at the different developmental stages without the Gal4 driver. As shown in Fig. 2a, the luciferase intensities from the pNP-luciferase transgenic flies are similar to those of the wild-type animals at all developmental stages analyzed, but their levels are all dramatically increased in pVALIUM20-luciferase animals after the embryonic stage. To test the potential tissue-specific differences, we dissected different tissues from the third instar larvae and performed the luciferase assays. Similar luciferase intensities were observed in the muscle and the central nervous system (brain and nerve cord) with both vectors (Fig. 2b). However, we observed the significantly elevated basal expression of luciferase in salivary gland, male gonad, female gonad, fat body, and wing disc from the pVALIUM20 system, being 685, 41, 42, 167, and 4 fold higher than in the corresponding tissues from the pNP system (Fig. 2b).

Significantly reduced leaky expression from basal promoter. a Relative luciferase expression levels at different developmental stages of pVALIUM20-luciferase and pNP-luciferase flies without the Gal4 driver (n = 5, mean ± s.d.). b Relative luciferase expression levels in different tissues of pVALIUM20-luciferase and pNP-luciferase flies at larval stage without the Gal4 driver (n = 3, mean ± s.d.). c Immunostaining of polytene chromosome from pVALIUM20-Hp1a and pNP-Hp1a without the induction of Gal4. DNA was visualized with DAPI and HP1a was stained with mouse monoclonal anti-HP1a (HP1a C1A9). Dotted circles indicate chromocenter. Scale bars, 20 μm. d Quantification of the relative HP1a intensity at chromocenter of polytene chromosome in pVALIUM20-Hp1a or pNP-Hp1a without the Gal4 driver (n = 10, mean ± s.d.). Data are evaluated with one-tailed Student’s t-test. e Relative expression of HP1a in control, pVALIUM20-Hp1a or pNP-Hp1a salivary gland without the driving of Gal4 (n = 3, mean ± s.d.). Data are evaluated using one-tailed Student’s t-test. f The heterozygous percentage of pVALIUM20 system based transgenic lines and pNP system based transgenic lines

To validate these observations based on the luciferase reporter, we compared the levels of heterochromatin protein 1 (HP1) by immunostaining using the polytene chromosomes from the pVALIUM20-Hp1a and pNP-Hp1a larvae without Gal4. Compared with the control and pNP-Hp1a, the levels of HP1a on polytene chromosomes from the pVALIUM20-Hp1a larvae were significantly decreased (Fig. 2c), and the relative HP1a intensity at the chromocenter was down to 20% (Fig. 2d), suggesting the leaky expression of shRNA in pVALIUM20-Hp1a. Consistent with this observation, we used the qRT-PCR assay and observed that the Hp1a mRNA levels in the salivary gland of pVALIUM20-Hp1a larvae were also significantly reduced compared to the control, while no differences in Hp1a mRNA levels were observed between pNP-Hp1a larvae and the control larvae at the same developmental stage (Fig. 2e). Taken together, these data support that, compared with the pVALIUM20 vector, the pNP system had significantly reduced the leaky expression.

To further test whether the leaky expression from the pVALIUM20 vector is deleterious to fly development thereby increasing the heterozygous rate of the transgenic animals, we built pVALIUM20 and pNP based transgenic lines against more than 300 genes, respectively (Supplementary Data 2). For each target gene, same shRNA was cloned into pVALIUM20 or pNP vector, respectively, and they were inserted in the same chromosome locus. When comparing the heterozygous rate of these transgenic lines, we observed that almost 15% of the pVALIUM20 system based transgenic lines were heterozygous, which is consistent with the rate in existing stock centers, while the pNP system based transgenic lines exhibited only 0.6% heterozygosity (Fig. 2f). These results demonstrate that the leaky expression is significantly reduced in the pNP system, also decreases the side effect shown by the pVALIUM20 system.

The pNP system is more efficient than the pVALIUM20 system

The pVALIUM20 system works well in somatic cells and female germline for many genes however, shRNAs targeting some of the functional genes in development generated no obvious or only weak phenotypes. To validate whether the pNP system is more efficient than the pVALIUM20 system, first we tested the expression of luciferase in different tissues of pNP-luciferase and pVALIUM20-luciferase transgenic flies using different Gal4 drivers, including elav-Gal4, MS1096-Gal4, Nub-Gal4, MTD-Gal4, nos-Gal4, GMR-Gal4, and esg-Gal4. As shown in Supplementary Fig. 3, both pNP-luciferase and pVALIUM20-luciferase transgenic flies showed highly expressed luciferase comparing with control. Furthermore, the luciferase intensities of pNP-luciferase transgenic flies are all significantly higher than pVALIUM20-luciferase animals using these drivers, indicating the pNP system is more efficient. Then we chose the same shRNAs, targeting genes encoding proteins known for their critical functions during the development of eye, wing or ovary, and generated transgenic lines using the pNP system. The genes targeted were E2F1, Upf1, aTub67C, and egg.

In our first example, we tested E2F1, the key transcription factor involved in regulating the G1-S phase transition of the cell cycle 27,28 . Depletion of E2F1 in the eye using ey-Gal4 in the pVALIUM20-E2F1 system slightly reduced eye size in contrast, the pNP-E2F1 system combined with ey-Gal4 generated a much smaller eye (Fig. 3a). In our second case study, we analyzed Upf1, an important protein involved in nonsense mRNA mediated decay 29 , since knocking down Upf1 using MS1096-Gal4 and the pVALIUM20-Upf1 system did not generate obvious phenotypes. However, we found that depletion of Upf1 using pNP-Upf1 driven by MS1096-Gal4 led to small wings with disrupted anterior cross vein (a-cv) (Fig. 3b). Similarly, depleting aTub67C in the wing using the pVALIUM20-aTub67C system and Nub-Gal4 failed to generate any phenotypes, presumably due to the high abundance of this transcript in cells. Nevertheless, a similar experiment using the pNP-aTub67C system led to extremely small wings (Fig. 3c). These results suggest that the pNP system produces stronger phenotypes than pVALIUM20 in somatic tissues.

The pNP system is superior to the pVALIUM20 system. a Knock down of E2F1 in the eye using the pVALIUM20 system or the pNP system with the same shRNA driven by ey-Gal4. Scale bars, 200 μm. b RNAi of Upf1 in the wing using MS1096-Gal4. Scale bars, 500 μm. c Targeting of aTub67C with the pVALIUM20 system or the pNP system driven by Nub-Gal4. Scale bars, 500 μm. d Knock down of egg in the germline controlled by nos-Gal4. Scale bars, 300 μm. e Fertility rates of control, pVALIUM20-egg and pNP-egg female flies using the nos-Gal4 driver (n = 10, mean ± s.d.). Data are evaluated with one-tailed Student’s t-test. f qRT-PCR analysis of the RNAi efficiency of E2F1, Upf1, aTub67C, and egg, respectively (n = 3, mean ± s.d.). Data are evaluated using one-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

To compare the knockdown efficiency of these two systems in the female germline, we crossed the nos-Gal4 line with pNP-egg and pVALIUM20-egg. As shown in Fig. 3d, the ovaries from pVALIUM20-egg females were smaller than controls, but the ovaries from the pNP-egg females were further reduced in size (Fig. 3d). Consequently, compared with a few eggs produced from the pVALIUM20-egg females, we did not obtain any eggs from the pNP-egg females (Fig. 3e). These results suggest that the pNP system also works more efficiently than the pVALIUM20 system in the female germline. Moreover, to exclude the possibility of off-target effect and rescue the phenotype generated in Fig. 3, we overexpressed the target genes that are insensitive to the hairpins in the RNAi flies driven by specific Gal4 lines. As shown in Supplementary Fig. 4, both the pVALIUM20-induced phenotype and the pNP-induced phenotypes were all significantly rescued, respectively, compared with the RNAi phenotypes alone, suggesting the specificity of these RNAi. Taken together, these results support the efficiency and specificity of this pNP system.

To further validate the notion that the pNP system is more efficient in depleting genes of interest than the pVALIUM20 system, we quantified the mRNA levels using the qRT-PCR assay. Although the pVALIUM20 system significantly reduced the transcripts of E2F1, Upf1, and egg compared to the control, greater depletion of these mRNAs was achieved sing the pNP system (Fig. 3f). For the aTub67C gene, the pVALIUM20 system did not affect mRNA level, but significant reduction was observed in wing discs using the pNP system (Fig. 3f). These observations are consistent with the aforementioned phenotypic assays. Remarkably, as a member of the tubulin family, aTub67C is highly expressed in cells the pVALIUM20 system failed to knock down aTub67C transcripts (Fig. 3f), implying the weakness of the pVALIUM20 system in depleting highly expressed genes. Taken together, these results suggest that the pNP system is more efficient than the pVALIUM20 system in both soma and female germline.

The pNP system efficiently modulates high expression genes

To efficiently deplete genes that are highly expressed in cells has been a challenge in the field. The observations we made with the aTub67C gene prompted us to test additional cases to compare the pNP system with the pVALIUM20 system. For this, we chose a set of multiple-copy genes, histone H1, H2A, H2B, H3, and H4, all highly expressed in cells. Using the same hairpins for each histone gene, we constructed transgenic RNAi flies based on the pNP system or the pVALIUM20 system. When driven by MS1096-Gal4, knock down of all the histones using the pNP system generated severe wing defects (Supplementary Fig. 5), consistent with the critical role of histones in cell viability. Surprisingly, depleting H1 or H3 using the pVALIUM20 system generated weak or no aberrant phenotypes, while knocking down H2A, H2B, or H4 in this system caused lethality before the pupal stage (Supplementary Fig. 5). Given that the pNP system is more efficient but less leaky than the pVALIUM20 system, we suspected that the leaky expression of pVALIUM20-H2A, H2B, and H4 plus the MS1096-Gal4 induced RNAi in early developmental stages may cause the lethality of these pVALIUM20 transgenic lines.

To test this idea, we selected the Nub-Gal4 tub-Gal80 ts system to compare the wing phenotypes using pNP and pVALIUM20. Gal80 ts is a temperature-sensitive repressor of Gal4, under the control of the tubulin promoter 30,31 . The crossed flies were first maintained under the permissive temperature 18 ℃ which allows Gal80 to repress Gal4 activities. The third instar larvae were then shifted to 29 ℃, which inactivates Gal80 thereby allowing Gal4-dependent expression to occur normally and deplete the histone genes. As shown in Fig. 4a, we observed the severely disrupted wing phenotype for all the histones depleted using the pNP system, similar to the RNAi phenotypes driven by MS1096-Gal4 (Supplementary Fig. 5). In contrast, the pVALIUM20 system showed no obvious phenotype for any of the depleted histones (Fig. 4a). The qRT-PCR result also confirmed that the pNP system was more efficient than the pVALIUM20 system (Fig. 4b), supporting the pNP system could efficiently modulate high expression genes with low false positive results.

The pNP system could efficiently modulate high expression genes. a The Nub-Gal4 tub-Gal80 ts system was used for conditional knock down of histones in the pNP and pVALIUM20 systems, with the flies first incubated at 18 °C and then shifted to 29 °C at third larval stage. Scale bars, 500 μm. b The act-Gal4 tub-Gal80 ts system was used for qRT-PCR analysis of the RNAi efficiency, targeting H1, H2A, H2B, H3, and H4 (n = 3, mean ± s.d.). Data are evaluated using one-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

The false positive results in pVALIUM20-H2A, H2B and H4 flies were likely from the combination of the leaky expression and MS1096-Gal4 induced RNAi in early developmental stage. In our analysis, pVALIUM20-H2A, H2B and H4 driven by MS1096-Gal4 were all lethal (Supplementary Fig. 5), but were viable and generated weak wing phenotypes using Nub-Gal4 tub-Gal80 ts , which prevented RNAi in early developmental stage (Fig. 4a). However, these hairpins driven by MS1096-Gal4 in the more efficient pNP system (also with low leaky expression) were viable (Supplementary Fig. 5) and gave the same severe phenotypes as Nub-Gal4 tub-Gal80 ts generated (Fig. 4a). Taken together, these results suggest that the leaky expression of the pVALIUM20 system plus MS1096-Gal4 induced RNAi in early developmental stage can cause false positive results.

These observations indicated that the pVALIUM20 system may cause lethality because of leaky and non-specific expression, which can be problematic particularly when applying this system for large-scale genetic screens. For example, we randomly selected 20 genes for which shRNAs in pNP gave wing defect phenotypes if crossed with wing-specific Gal4 three of them were lethal if the same hairpins were used in pVALIUM20, further supporting the high tissue specificity of the pNP system (Supplementary Data 3). Taken together, these results show that the pNP system is more efficient than the pVALIUM20 system in knocking down genes of interest, including those with high levels of expression, and that it produces the expected strong phenotypes without obvious leaky expression that often observed in the pVALIUM20 system.

The pNP system can simultaneously target multiple genes

Proteins encoded by two or more genes in Drosophila can have partially overlapping or redundant functions, and thus depleting one of them may result in little or no effect on functional or phenotypic analyses. In addition, depletion of a single subunit of a protein complex may result in the formation of partial protein complexes, which can have dominant-negative or neomorphic effects in vivo. Thus it is advantageous to simultaneously deplete multiple genes. However, existing transgenic RNAi vectors can only accept a single shRNA therefore, to simultaneously target two or more genes cumbersome genetic crosses are required. Another problem is that genetic recombination may reduce RNAi efficiency because of Gal4 dilution, as well as that unhealthy recombinants frequently occur.

An intergenic linker region between miR-2a-1 and miR-2b-2 was inserted into the pNP vector following the miR1 scaffold (Supplementary Fig. 1), which could express multiple shRNAs. We performed three sets of experiments in parallel, comparing the efficiency to deplete these sets of target genes in the same vector individually or simultaneously. First, we subcloned a hairpin targeting Notch gene and a hairpin targeting white gene into the pNP vector, designated as the pNP-N-W vector, and generated transgenic lines. Same hairpins subcloned into the pNP vector individually were used as positive controls. When driven by the eye-specific GMR-Gal4 line, RNAi depletion of white completely eliminated red pigments in the eyes, while knocking down either Notch alone or Notch and white together (Notch-white) caused lethality at larval stage (Fig. 5a). If RNAi was induced by the wing-specific C96-Gal4 line, depleting white in the wing did not generate any abnormal phenotypes as expected, whereas depletion of either Notch alone or Notch-white generated the same wing phenotype with reduced size. The observation that the effects on the eye by depleting the white gene and the wing effects caused by knocking down Notch occur independently within the pNP-N-W line demonstrates that the pNP vector can simultaneously target multiple genes with one RNAi vector (Fig. 5b). To test the possibility that the RNAi-induced phenotypes might be affected by the order of the hairpins in the pNP vector, we generated a transgenic line with the white hairpin in front of the Notch hairpin (pNP-W-N), and then compared the RNAi efficiency with that of the pNP-N-W line. As shown in Fig. 5a, b, both the pNP-W-N and pNP-N-W lines generated the identical phenotypes when driven by GMR-Gal4 or C96-Gal4 lines, suggesting that the hairpins in the pNP vector function independently, without a sequential effect.

The pNP system can simultaneously target multiple genes. a RNAi against white caused complete loss of red pigment in eye also note that knock down of notch in eye using GMR-Gal4 caused lethality. Scale bars, 200 μm. b Compared with control, pNP-N, pNP-W-N and pNP-N-W driven by C96-Gal4 all showed similar Notch wing defect phenotypes. Scale bars, 500 μm. c Disruption of Ci driven by Nub-Gal4 produced anterior fusion of L3 and L4 veins of the wing, while knock down of E2F1 caused small wing with anterior fusion of L2 and L3 veins. The combined phenotype of anterior fusion of L2, L3, L4 veins and small wing showed in pNP-Ci-E2F1 and pNP-E2F1-Ci flies. Scale bars, 500 μm. d, e Traditional genetic combination of pNP-Ci and pNP-E2F1 exhibited a much weaker phenotype. Only a small proportion of pNP-Ci pNP-E2F1 flies showed both Ci KD and E2F1 KD phenotype most of them generated either Ci KD or E2F1 KD, while there were even some similar with wild-type phenotype. Scale bars, 500 μm

In addition, we chose another set of protein coding genes, Ci and E2F1, as depleting both of them using Nub-Gal4 generated distinct phenotypes in the wing. As shown in Fig. 5c, depleting Ci using pNP-Ci generated anterior fusion between L3 and L4 without affecting the size of the wing, while knocking down E2F1 using pNP-E2F1 led to a smaller wing with vein fusion between L2 and L3. When both genes were depleted using either the pNP-Ci-E2F1 line or the pNP-E2F1-Ci line, we observed the same small-sized wing as for pNP-E2F1, which was also carrying the vein fusions one was between L2 and L3 from E2F1 KD, another was between L3 and L4 from Ci KD (Fig. 5c). These phenotypes appear to be due to the additive effect of those caused by depleting either E2F1 or Ci alone, further supporting the high efficiency of the pNP system in targeting multiple genes. Again, no sequential effect was observed by comparing the pNP-Ci-E2F1 line and the pNP-E2F1-Ci line (Fig. 5c). To compare the efficiency of multiple shRNA knock down from the pNP system with traditional genetic recombination, pNP-Ci and pNP-E2F1 were genetically recombined first and then crossed with the Nub-Gal4 line. Interestingly, only 2.47% of the pNP-Ci pNP-E2F1 flies showed both Ci KD and E2F1 KD phenotypes most of them generated either the Ci KD or E2F1 KD phenotype alone, while 6.81% in fact produced no obvious phenotype (Fig. 5d, e). This variable result illustrates another drawback of using traditional genetic recombination to knock down multiple genes, presumably due to competition for Gal4 binding by the two UAS cassettes. Moreover, the flies with the integrated line of pNP-Ci pNP-E2F1 appeared weak as homozygosity was lethal before adulthood, likely due to too many exogenous genetic components. These observations further support the notion that the pNP system is more robust and versatile than traditional recombination to target multiple genes.

To further validate the ability of pNP in depleting multiple genes simultaneously, we applied this system to Polycomb repressive complex 1 (PRC1), which function as transcriptional repressor complexes essential for normal development 32 . Simultaneous knockdown of four core proteins of PRC1 generated severe eye defects, while depleting of them individually did not cause any defects in the eye (Supplementary Fig. 6a). The RNAi efficiency of tetradic-gene-KD is also comparable with single-gene -KD tested by qRT-PCR assay (Supplementary Fig. 6b). Taken together, these results demonstrate the remarkable effectiveness of the pNP system to simultaneously knock down multiple genes.

HATs regulate eye development through Wnt signaling pathways

In an effort to test this transgenic RNAi system for studying proteins with partially redundant functions, we chose to study a family of histone acetyltransferases (HATs), the enzymes that mainly acetylate lysine residuals on histones. Given that the HAT family of enzymes regulate transcriptional activation, DNA repair, and the cell cycle, dysregulation of their activities is often linked to epigenetic alterations in many developmental disorders and diseases such as cancer 33 . Transcriptional activation requires multiple HATs, and the functions of some HATs are generally redundant thus the depletion of one HAT is insufficient to produce phenotypes. We focused our analyses on a set of HATs belonged to the MYST or GNAT families, including chm, Tip60, and Gcn5. After generating the transgenic RNAi lines against chm, Tip60, Gcn5, and the combinations of chm-Tip60 and chm-Tip60-Gcn5, we crossed these lines with the eye-specific GMR-Gal4 line. We observed that the pNP-chm, pNP-Tip60, pNP-Gcn5, and pNP-chm-Tip60 did not produce any defects in the eye (Fig. 6a). However, simultaneously depleting all three HATs in the eye using the pNP-chm-Tip60-Gcn5 line led to a severely defective eye phenotype, characterized by the overproduction of eye pigment, necrotic cell death, and slightly reduced eye size (Fig. 6a) This is unlikely to be caused by off-target effects, as depletion of any one of them produced no obvious defects.

Histone acetyltransferases regulate eye development through Wnt signaling pathways. a chm, Tip60, Gcn5 single knock down or chm-Tip60 double knock down exhibited wild-type phenotype driven by GMR-Gal4, while simultaneous knock down of chm-Tip60-Gcn5 using the pNP system produced severe eye defects. Scale bars, 200 μm. b qRT-PCR results show that chm, Tip60, Gcn5 are all reduced to comparable level in single gene knockdown or triple genes knockdown flies. (n = 3, mean ± s.d.). c Wnt signaling pathway was significantly up-regulated as represented by Wg via qRT-PCR assay (n = 3, mean ± s.d.). Data are evaluated using one-tailed Student’s t-test. d Immunostaining result showed up-regulated Wg in GFP-marked chm-Tip60-Gcn5 KD clones. Dotted lines showed the GFP marked clone. Compared with control (indicated by long tail arrows), Wg signals were significantly increased in chm-Tip60-Gcn5 KD clone (indicated by short tail arrows). Scale bars, 50 μm. e Transcriptional activation of wg produced similar eye phenotype as chm-Tip60-Gcn5 KD flies. Knockdown of wg or arm at the background of chm-Tip60-Gcn5 triple RNAi clearly rescued the eye phenotype. Scale bars, 200 μm

To confirm that this phenotype is indeed caused by depletion of chm, Tip60, and Gcn5, we performed a qRT-PCR assay to measure the transcripts of these genes in eye discs after RNAi triggered by GMR-Gal4. Compared with the control, the transcripts from individual RNAi lines and the triple RNAi line were all significantly reduced (Fig. 6b). Notably, the efficiency of RNAi depletion in the triple RNAi line was comparable to that of the individual RNAi lines, further demonstrating the power of the pNP system to simultaneously target multiple genes. To further exclude the possibility of off-target effects, we overexpressed chm-T2A-Tip60-T2A-Gcn5 that insensitive to the hairpins by using the T2A self-cleaving peptide in the chm-Tip60-Gcn5 triple KD flies, and the KD phenotype was fully rescued (Supplementary Fig. 7b), supporting the specificity of this RNAi experiment. Furthermore, we also generated a transgenic activation line that targeting these three genes simultaneously using the flySAM system we developed recently 34 . As shown in Supplementary Fig. 7c, activation of these three genes also significantly rescued chm-Tip60-Gcn5 triple KD tumor-like phenotype, further supporting the specificity of this triple KD result.

Furthermore, to validate the enzyme activity of chm, Tip60, Gcn5 on histone acetylation, we performed immunostaining to detect the level of global H4 acetylation in polytene chromosomes after RNAi induced by salivary gland-specific 1824-Gal4. The intensity of immunostaining against H4 acetylation in polytene chromosomes from the pNP-chm, pNP-Tip60, pNP-Gcn5 lines was slightly lower compared to the control, whereas the levels of H4 acetylation in polytene chromosomes from the pNP-chm-Tip60-Gcn5 line were almost undetectable (Supplementary Fig. 8). Taken together, these observations demonstrate the redundant roles of these HATs on epigenome, further supporting the ability of the pNP system to successfully modulate multiple genes.

To explore the molecular mechanism of the severe eye defect phenotype produced by triple RNAi against chm-Tip60-Gcn5, we detected the transcriptional level of genes downstream of several major signaling pathways, such as the JAK-STAT, EGFR, Hippo, JNK, dpp, Notch, and Wnt signaling pathways, because of their roles in regulating eye development. Specifically, we analyzed the expression of SOCS36E, aos, Yki, puc, dad, Su(H) and wg as the readouts for the activities of the aforementioned signaling pathways in eye discs with these HATs being depleted 35 . As shown in Fig. 6c, wg, a target of Wnt signaling, was significantly up-regulated by more than five-fold of the control, while the representative target genes of the other signaling pathways tested were not significantly affected in HAT-depleted eye discs. These observations indicate that Wnt may be the major signaling pathway being affected by these HATs in eye discs.

To test the effects of depletion of the HATs on Wnt signaling, we generated the chm-Tip60-Gcn5 KD clones marked with GFP in wing discs and then analyzed the levels of Wg expression using immunostaining. As shown in Fig. 6d, the chm-Tip60-Gcn5 KD clones showed clearly elevated levels of Wg proteins. Meanwhile, we constructed wg transcriptional activation flies using the CRISPRa system 34 , which showed exactly similar eye phenotype as the chm-Tip60-Gcn5 KD when driven by GMR-Gal4 (Fig. 6e). To further validate that the defective eye phenotype was indeed caused by elevated Wg signaling, we depleted wg in addition to the triple knocking down of chm, Tip60 and Gcn5. Compared to the chm-Tip60-Gcn5-GFP-RNAi control, which was the same as the chm-Tip60-Gcn5 KD phenotype, we observed that knocking down wg significantly rescued the eye phenotype caused by depletion of HATs. Similarly, simultaneous depletion of the three HATs and arm, encoding a key component of the Wg signaling pathway, dramatically rescued the defective eye phenotype caused by depletion of these HATs (Fig. 6e). Taken together, these analyses suggest that chm, Tip60 and Gcn5 synergistically play critical roles in regulating eye development, mainly through modulating the Wnt signaling pathway.


Phenotypic Characterization of Transgenic Miscanthus sinensis Plants Overexpressing Arabidopsis Phytochrome B

Phytochromes are dimeric pigment proteins with reversible photochromism between red and far-red light-absorbing forms. They are photoreceptors that regulate various aspects of plant growth and development and have been used for biotechnological applications to improve agricultural performance of crops. Miscanthus species have been suggested as one of the most promising energy crops. In this paper, Arabidopsis phytochrome B (PHYB) gene was introduced into Miscanthus sinensis using Agrobacterium-mediated transformation method that we developed recently, with the herbicide resistance gene (BAR) as a selection marker. After putative transgenic plants were selected using the herbicide resistance assay, genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by Northern blot analysis. Compared to nontransformed control plants, transgenic plants overexpressing PHYB showed phenotypes with increased phytochrome B function, which includes increased chlorophyll content, decreased plant height, and delayed flowering. Therefore, these results suggest that Arabidopsis phytochrome B is functional in M. sinensis and provide a method to develop Miscanthus varieties with enhanced agricultural performance using phytochromes.

1. Introduction

Phytochromes are photoreceptors that regulate various aspects of plant growth and development in response to red and far-red light signals from the environment [1, 2]. They are dimeric chromopeptides with each monomer (120–130 kDa) possessing a covalently linked open-tetrapyrrole chromophore called phytochromobilin via a thioether linkage to a cysteine residue [3]. The most important characteristic of phytochromes is its reversible photochromism: the property of changing color on photon absorption and of reverting to the original form on the absorption of another photon. Phytochromes are synthesized in red light-absorbing Pr form (

nm) which can be phototransformed into far-red light-absorbing Pfr form (

nm) upon exposure to red light. The functional activities of the phytochromes are modulated by the photochromic transformation between these two forms [4, 5]. Generally, the Pr-to-Pfr phototransformation of phytochrome is known to induce the highly regulated signaling network for photomorphogenesis in plants [6–8].

Phytochrome-mediated photomorphogenic responses include germination, stem and leaf growth, chloroplast development, biosynthesis of chlorophylls and other pigments, shade avoidance, circadian rhythm, and flowering [9]. In the aspect of biotechnological applications of phytochromes, physiological analysis of plants overexpressing phytochrome transgenes has previously revealed the potential of transgenic approaches to modifying the architecture of crop plants [10]. As an example, tobacco plants expressing oat phytochrome A transgene (PHYA) exhibit proximity-conditional dwarfing which leads to up to 20% improvement in harvest index [11]. In principle, phytochromes mediate plant shade avoidance responses in which plants react to far-red radiation reflected from neighbors and induce elongation growth to prevent overtopping by competitors growing alongside [12]. If the shade avoidance responses are induced, the reallocation of resources into elongation growth might result in reduced leaf and storage organ production with a decrease in crop yield [13]. Thus, the shade avoidance responses can be detrimental in agriculture because it limits the planting density. Therefore, it has suggested that plant productivity can be enhanced by reducing perception of the neighbors (i.e., suppression of shade avoidance responses) through the manipulation of phytochrome red/far-red light perception [14].

Miscanthus species are tall perennial rhizomatous grasses with C4 photosynthesis which tend to give high biomass yields annually over a wide range of climates [15, 16]. Thus, they are suggested as one of the most promising energy crops for biomass production [17]. So far, the triploid hybrid Miscanthus × giganteus between the diploid M. sinensis and the tetraploid M. sacchariflorus is currently the commercially grown species in the genus [18, 19]. However, this hybrid is sterile and lacks genetic variation, so the necessity for seed-propagated varieties in genetically stable and fertile species, such as M. sinensis, has been raised [20].

The genetic improvement by conventional breeding has been used to improve traits of many species, but its success has been limited by barriers such as sexual reproduction and the relatively long time periods required for breeding programs. More recently, genetic engineering methods with the help of plant transformation technologies have been used to improve many species more efficiently, whereby useful traits have been introduced from a broader range of sources within an economically viable time frame [21]. In the case of Miscanthus species, the genetic transformation systems have been established very recently using particle bombardment-mediated and Agrobacterium-mediated methods [22, 23]. However, there is no report thus far of transgenic Miscanthus plants with a useful gene other than selective marker genes. Therefore, genetically engineered Miscanthus plants need to be further developed by introducing a useful gene using the established transformation method.

The objective of this study was to develop transgenic M. sinensis plants overexpressing Arabidopsis phytochrome B gene (PHYB) using the Agrobacterium-mediated genetic transformation method that we developed recently. In addition, the phenotypes of the transgenic plants were investigated in terms of phytochrome function, which includes chlorophyll content, plant height, and flowering time. In the present study, we successfully obtained transgenic M. sinensis plants with a vector harboring PHYB as well as the herbicide resistance gene (BAR) as a selection marker and found that the overexpression of PHYB increased chlorophyll content, reduced plant height, and delayed flowering in M. sinensis plants. Therefore, the present work provides a method for the development of genetically engineered Miscanthus varieties with enhanced agricultural performance using phytochromes.

2. Materials and Methods

2.1. Preparation of Recombinant Arabidopsis Phytochrome B Proteins

Chromophore-assembled holoproteins of Arabidopsis phytochrome B (phyB) were expressed, reconstituted, and purified as previously described [24, 25]. Full-length Arabidopsis PHYB gene was subcloned into a Pichia expression vector pPIC3.5K (Invitrogen). Ten amino acid streptavidin affinity tag from pASK75 vector (Biometra) was attached to the 3′ end of the PHYB gene. The primers 5′-CTCCCCGGGTACGTAACCATGGTTTCCGGAGTCGGGG-3′ (forward, SmaI/SnaBI) and 5′-TCGCAGCGCTATATGGCATCATCAGCATCATG-3′ (reverse, Eco47III) were used for the subcloning. The pPIC3.5K construct bearing PHYB was then transformed into Pichia cells by means of a Micropulser Electroporation apparatus (Bio-Rad). Recombinant phytochrome proteins were expressed in the Pichia expression system (Invitrogen) and purified by streptavidin affinity chromatography (IBA), according to the procedure described by the manufacturer. Phycocyanobilin (PCB) was prepared from Spirulina platensis extracts by methanolysis as described [25] and used as chromophores for the holo-phytochrome assembly. Crude extracts were prepared by breaking Pichia cells in liquid nitrogen using a homogenizer (Nihonseiki Kaisha). The phytochrome samples were then precipitated by adding 0.23 g/L ammonium sulfate and resuspended in a buffer (100 mM Tris, pH 7.8, 1 mM EDTA), the PCB chromophores in DMSO were added to the samples at a final concentration of 10 μM, and the mixture was incubated for in vitro reconstitution on ice for 1 h. After dialysis to remove free chromophores, the samples were loaded onto affinity chromatography columns and holo-phyB proteins were purified.

2.2. Zn 2+ Fluorescence and Spectroscopic Analysis

For Zn 2+ fluorescence assay to assess the chromophore ligation, the protein samples were analyzed on a 10% SDS-PAGE gel and the gel was soaked in 20 mM zinc acetate/150 mM Tris-HCl (pH 7.0) for 5–30 min at room temperature with gentle shaking. Zinc fluorescence of holo-phytochromes was visualized under UV light (312 nm). For the spectroscopic analysis of recombinant phyB, absorption spectra were recorded by a diode array UV/VIS spectrophotometer (Cary) after red or far-red light irradiation. All the spectroscopic experiments were carried out under the green safety light condition, which consisted of a white fluorescent lamp equipped with a plastic filter (Rosco) with a maximal transmittance at 500 nm, and a fiber optic illuminator system (Cole-Parmer) equipped with 656 and 730 nm interference filters (Oriel) was used as a light source. The light intensity was 8 W/m 2 for red light and 6 W/m 2 for far-red light. The samples were illuminated with red for 15 min or far-red light for 10 min. A difference spectrum was obtained by subtracting the Pfr spectrum from the Pr spectrum and used to determine the interpeak distance between Pr and Pfr absorption wavelength maxima (

of maxima) and the ratio of Pr and Pfr absorbance peaks (APfr/APr).

2.3. Plasmid and Agrobacterium Strain Used for Genetic Transformation

Arabidopsis PHYB gene (AT2G18790) was subcloned into the binary vector pCAMBIA3301 with BamHI/SmaI under the control of maize ubiquitin promoter (Pubi) and Agrobacterium tumefaciens NOS gene terminator. pCAMBIA3301 carries BAR gene for herbicide resistance as a selectable marker, and the BAR gene encodes a phosphinotricin acetyltransferase that confers resistance to the herbicide, phosphinotricin (PPT). The binary vector DNA was then used to introduce into A. tumefaciens strain EHA105 by the freeze-thaw method [26].

2.4. Generation of Putative Transgenic M. sinensis Plants

SNU-M-045 germplasm seeds of M. sinensis that have been maintained in Seoul National University were used for the transformation, and tissue culture and genetic transformation of M. sinensis were performed as described [23]. Mature seeds-derived embryogenic calli and A. tumefaciens EHA105 harboring pCAMBIA3301 with PHYB were used for the genetic transformation. Embryogenic calli were induced and cultured on the callus induction medium (MS salts and vitamins, 3% sucrose, 3 mg/L 2,4-D, 750 mg/L MgCl2·6H2O, 25 mM L-proline, 0.2 g/L Gelrite, pH5.7) for 8 to 10 weeks. The embryogenic calli were then immersed in Agrobacterium suspensions and incubated for 15 min with gentle shaking followed by removal of excess bacteria and air-drying on filter paper. Infected calli were then transferred onto cocultivation medium (MS salts and vitamins, 2% sucrose, 1% glucose, 3 mg/L 2,4-D, 400 μM acetosyringone, 3 g/L Gelrite, pH 5.7) and incubated in the dark at 25°C for 5 days. During the transformation process, GUS staining assays were performed to check whether or not the embryogenic calli were transformed. After transformation, transgenic calli were selected on the selection medium containing 5 mg/L PPT, and transgenic shoots were induced on the regeneration medium with 3 mg/L PPT. Plantlets with well-developed roots were established in soil and grown for 2 weeks, prior to herbicide treatment. Herbicide resistance assay was then performed by spraying 0.4% (v/v) BASTA (which contains 18% glufosinate ammonium), and the herbicide resistance of putative transgenic plants was determined after 14 days.

2.5. Molecular Analyses of Transgenic M. sinensis Plants

Genomic PCR, Southern blot, and Northern blot analyses of transgenic plants were performed as previously described [27, 28]. For genomic PCR analysis, total genomic DNA was isolated from the leaves of greenhouse-grown plants, and the coding region for the PHYB or BAR transgene was amplified by PCR from either genomic DNA or a positive-control vector, using the following sets of oligonucleotide primers: 5′-TCGGCGTTCCGGTCCATGGTTAG-3′ (forward) and 5′-GCAGCGCTCAGTGTCTGCGTTCTCAAAACG-3′ (reverse) for PHYB, 5′-CTACCATGAGCCCAGAACGACG-3′ (forward) and 5′-CTGCCAGAAACCCACGTCATGCCAGTTC-3′ (reverse) for BAR. The actin gene (ACT) of M. sinensis was also amplified using the same template and the primers 5′-AACTGGGATGATATGGAGAA-3′ (forward) and 5′-CCTCCAATCCAGACACTGTA-3′ (reverse) and then run as a loading control of genomic DNA. The PCR products of PHYB, BAR, and ACT were expected to be 1106, 421, and 1,046 bp, respectively.

For the Southern blot analysis, the genomic DNA was digested with either HindIII or EcoRI, and hybridizations were carried out with the BAR gene probe that was labeled with [α 32 P] dCTP using the Radiprime II Random Prime Labeling System (Amersham Biosciences). For Northern blot analysis, total RNA was extracted from leaves using Trizol reagent (Invitrogen), and hybridizations were carried out with [α 32 P] dCTP-labeled PHYB probe.

2.6. Measurement of Chlorophyll Content

Chlorophyll (chl) content was measured with the third leaf of the tillers harvested on DOY (day of year) 273. The leaves were extracted with 80% acetone and the solution absorbance was measured at 645 nm (

). Total chlorophyll content was calculated from the equation: total chlorophyll (μg/mL)

. The relative chlorophyll content was then calculated by setting the chlorophyll content of nontransgenic control plant to 100%.

2.7. Phenotypic Analyses of Transgenic M. sinensis Plants

M. sinensis plants in soil were grown and vegetatively propagated in a culture room (24–26°C with a 16 h photoperiod). For the investigation of phenotypes, the plants were then transferred and planted from the culture room onto an isolated LMO greenhouse of Seoul National University in Suwon, Korea (N 37° 16′ 15.08′′, E 126° 59′ 22.02′′). Plant heights were measured as the lengths from the ground to the top of each plant. Heading and flowering dates were defined as the emergence of the panicle tip from the flag leaf sheath and the opening of the first flower, respectively.

2.8. Statistical Analysis

The results of the physiological parameters were analyzed using ANOVA with IBM SPSS statistics 20 software. Significant difference from the control value(s) was determined at

level. All of the data represented the mean ± SD or SE of at least three independent experiments.

3. Results and Discussion

3.1. Photochemical Properties of Arabidopsis phyB

There are five isoforms of plant phytochromes (phyA–phyE) in the dicot model plant Arabidopsis thaliana, among which phyA and phyB have been known to play major roles in plants [29, 30]. For example, phyA regulates seed germination and seedling growth in response to far-red light (FR), whereas phyB regulates seed germination in response to red light (R), plant growth and shade avoidance in response to R/FR ratio, and flowering time [4]. Especially, it has been well known that phyB-deficient mutant (hy3) shows phenotypes of constitutive shade avoidance and early flowering [31], suggesting that phyB is necessary to suppress the shade avoidance responses and flowering in plants, which is necessary to improve plant productivity. Therefore, we planned to introduce Arabidopsis phyB into M. sinensis and to investigate the phenotypes of the monocot crop overexpressing dicot phyB.

Before the genetic transformation, we first performed the photochemical analysis of Arabidopsis phyB using purified recombinant proteins, because phyB has not been characterized well due to its low abundance in native tissue [32]. In contrast, the spectral properties of phyA have been well characterized because of the relatively high abundance of this photoreceptor in dark-grown tissue (

100 times more than phyB). In the present study, we expressed and purified recombinant phyB proteins successfully using the Pichia protein expression system and streptavidin affinity chromatography (Figure 1(a)). Purified Arabidopsis phyB showed ligation with the chromophore phycocyanobilin (PCB) and normal patterns of absorbance and difference spectra (Figure 1). Spectroscopic analysis of the purified phyB proteins revealed that the absorbance wavelength maxima of Pr (

) forms are 650 nm and 714 nm, respectively. Compared with the photochemical properties of oat phyA in a previous report [25], Arabidopsis phyB showed similar properties to phyA including of maxima (64 m) and APfr/APr (1.09) obtained from the difference spectrum. The only observed difference was that the absorbance wavelengths of phyB were slightly blue-shifted (4–6 nm) compared with the absorbance peaks of oat phyA (654 nm for Pr and 720 nm for Pfr). Collectively, the results demonstrate that PCB-assembled recombinant Arabidopsis phyB has a typical photochromism of plant phytochromes.


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(b)
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DISCUSSION

A plethora of specific phenotypes induced by miRNA gain-of-function in vivo

There is abundant evidence from cell-based studies that animal miRNAs directly but mildly repress hundreds of targets (Lim et al., 2005 Hendrickson et al., 2009 Guo et al., 2010), even when measured in contexts of ectopic activity. Given this, the in vivo consequences of miRNA deregulation might reasonably have been supposed to often be subtle (with the view that few targets can be sufficiently suppressed to reveal loss-of-function phenotypes), or might often compromise general cell viability (with the view that the coordinate downregulation of hundreds of targets might cause cells to simply become unhealthy).

We describe a systematic in vivo examination of the consequences of targeted miRNA misexpression within the intact animal. In contrast to the aforementioned possibilities, we find that the majority of miRNAs tested generated diverse and relatively distinct mutant phenotypes, most of which could not be anticipated from target predictions or from the general fine-tuning model of miRNA function. Although a number of miRNAs have profoundly adverse consequences that might be due to cellular toxicity, many miRNA-induced phenotypes closely resemble those exhibited by mutants of genes in signaling/proliferation/apoptosis pathways that are crucial to tissue development and patterning. The present studies extend our earlier functional screens in cultured cells that linked miR-315 to activation of the Wnt pathway (Silver et al., 2007), and now permit diverse functional screening in the animal. Indeed, many miRNAs of unrelated sequences generated similar phenotypes in vivo, which might be explained if they hit different nodal points in the same pathways.

Many, if not most, genes contain conserved binding sites for multiple miRNAs. But it is clear that the simple presence of conserved miRNA-binding sites does not guarantee responsiveness in directed sensor assays. It is even more so the case that presence of cognate binding sites does not render a miRNA likely to be able to induce a corresponding loss-of-function phenotype in the animal, even when misexpressed (Silver et al., 2007). Even when using artificial shRNA constructs designed to have perfect complementarity for maximal effect, it is typical for them to elicit only partial knockdown or sometimes to not work at all. The known dose sensitivity of the core cell signaling pathways and patterning genes provides a genetic rationale for why they may be especially prone to be affected by miRNAs in a way that translates into overt mutant phenotypes (Hagen and Lai, 2008 Smibert and Lai, 2010). Such genetic connections can guide functional studies and point to likely target pathways, even when knowledge of relevant computationally predicted targets is lacking.

Beyond understanding the underlying genetic circuitry of insects, our studies highlight that ectopic miRNAs can generate specific developmental phenotypes, often as a result of altering tissue patterning, proliferation of apoptosis. This has substantial consequences for interpreting the etiology of disease and cancer. For example, the overexpression of a growing number of mammalian miRNAs can generate cell specification or metabolic defects, and miRNAs such as mir-21 (Medina et al., 2010) and mir-17-92 (He et al., 2005) are overt oncogenes. Our systematic screening in Drosophila strongly suggests that scores of vertebrate miRNAs may prove to induce relatively specific phenotypes in the animal, but that these may only rarely be predicted on the basis of computationally derived target associations.

A genetic resource for miRNA screening in vivo

A great deal of effort has been devoted to expanding collections of conditionally activated transgene insertions in Drosophila (Brand and Perrimon, 1993 Rorth et al., 1998). Over the past 15 years, these have been of tremendous use in revealing the biological activity and function of protein-coding genes. Here, we describe genome-wide collections of miRNA transgenes, and demonstrate their collectively diverse activities during wing development. These collections include both P insertion and attP insertion lines, providing a great deal of flexibility for their subsequent screening. The latter permits the activity of different miRNAs to be compared directly, whereas the former provides in many cases allelic series of transgene strengths. The availability of these lines permits a wide variety of screens using tissue- or cell-specific drivers, to evaluate the consequences of miRNA deregulation on development, as well as adult roles in physiology or behavior. Knowledge of their functional capacities can then inform the study of different miRNAs within their endogenous expression domains (Aboobaker et al., 2005 Berezikov et al., 2011).

While this work was under review, Cohen and colleagues described a smaller set of UAS-miRNA transgenes and their application towards searching for modifiers of a bristle phenotype of the cell cycle regulator minus (Szuplewski et al., 2012). Beyond a limited set of bristle modifiers, however, their analysis primarily revealed lethality as the outcome of miRNA expression (Szuplewski et al., 2012). We find over 100 of our miRNA transgenes induced lethality when broadly expressed with da-Gal4. However, our detailed analysis using a panel of wing drivers revealed a cornucopia of distinct phenotypes, many of which phenocopy the modulation of fundamental signaling pathways and patterning factors (Figs 2, 3, 4, 5 and 6). Our UAS-miRNA collections complement and substantially extend their transgenes, and together they constitute a formidable resource for in vivo analysis of miRNA activity. Many miRNAs have subtle if not undetectable loss-of-function phenotypes (Miska et al., 2007 Alvarez-Saavedra and Horvitz, 2010), but it is also the case that many miRNA mutants produce synthetic phenotypes in combination with other genetic insults (Brenner et al., 2010). Data such as ours provide a genetic basis for pursuing more than 100 demonstrable miRNA activities and many tens of compelling miRNA-target/pathway linkages, and can inform more complex interaction studies with miRNA sponges (Loya et al., 2009). Indeed, we provide proof of principle for how K box miRNA gain of function, which inhibited Notch signaling, informed sensitized genetic assays that revealed the endogenous activity of a likely highly redundant set of endogenous K box miRNAs in restricting Notch signaling during wing development. Our extensive assays provide compelling evidence of the usefulness of these genome-wide collections of conditionally activatable miRNA transgenes, and suggest that these may be well complemented by similar collections of miRNA sponge transgenes.


Conclusions

CRISPR-cas9 technology has been successfully used to knock-down several protein coding genes in several plant species. Although successful editing of non-coding RNAs has been demonstrated in animals [reviewed in Basak and Nithin (2015)], the only report on editing of a non-coding RNA is in tomato (Li et al. 2018). We demonstrate successful gene editing of a non-coding regulatory RNA TAS4a/b in grape cultivar 101-14, an anthocyanin producing rootstock. We further demonstrate fortuitous off-target effects of TAS4b guide RNA on TAS4a locus resulting in a chimeric TAS4a-b locus subject to homologous recombination events associated with off-target editing. Future studies are now possible to test the roles of Vvi-MYBA7 and Vvi-TAS4a/b/c in tissue-specific anthocyanin expression and the genes’ roles in microbe and virus disease etiologies and possibly feeding preferences of arthropod vectors (Zeilinger et al. 2018).


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Discussion

CRISPR/Cas9 specificity is affected by various factors, including features of off-target sites and effective concentrations of the Cas9/sgRNA complexes. Determination of CRISPR/Cas9 specificity also depends on assay methods, either biased or unbiased, and in silico off-target prediction algorithms. This complexity is regarded as a confounding factor for off-target mutation assays and the use of different standards for measuring and reporting off-target activity affects the preciseness of conclusions (Tsai and Joung 2016 Tycko et al. 2016 Wu et al. 2014).

CRISPR/Cas9 is known to be significantly more specific in plant cells than in human cells (Feng et al. 2014 Peterson et al. 2016 Wolt et al. 2016 Zhang et al. 2014). Two reports based on whole genome sequencing (WGS) or deep sequencing provide evidence that the editing efficiency of the CRISPR/Cas9 system is highly specific in Arabidopsis (Feng et al. 2014 Peterson et al. 2016). In one of these reports, deep sequencing of a total of 178 off-target sites demonstrated that the high specificity of CRISPR/Cas9 in Arabidopsis and the low expression levels of the Cas9 driven by the UBQ10 promoter were hypothesized to be the reason for the undetectable off-target events (Peterson et al. 2016). Actually, competitive binding of 14 sgRNA variants to Cas9 definitely led to significantly lower effective concentrations of each Cas9/sgRNA complex variant. Because out of the 14 sgRNAs, two (CLE18_2 and GLV8_1) did not show any evidence of on-target editing, the 26 off-target sites from these two sgRNAs could be excluded from the list of off-target sites. Thus, out of the rest of the 152 off-target sites from the 12 sgRNAs, 83% (126/152) and 17% (26/152) harbored 4 and 3 mismatches, respectively, indicating that the 12 sgRNAs were predicted to be specific by in silico analysis (Peterson et al. 2016). Therefore, lower effective concentrations of each Cas9/sgRNA complex variant and relatively specific sgRNAs might be the reasons for the reported high specificity of CRISPR/Cas9 in Arabidopsis.

In the other report, in-depth WGS of two T1 plants (#T1-46 and #T1-55) and one T2 plant (#T2-46) harboring GAI-sgRNA1 showed no indication of any off-target events in the potential off-target sites harboring 1–4 mismatches (Feng et al. 2014). However, although WGS is ideal for off-target mutation assays of individual plants, due to its high cost, it is not practical to systematically assess a large number of plants and sgRNA variants to determine Cas9 specificity (Wu et al. 2014). Thus, most low-frequency off-target events would go unaccounted (Wu et al. 2014). Although approximately 60 T1 plants harboring GAI-sgRNA1 were examined for off-target mutations by PCR followed by sequencing, a large number of T2 plants were not examined. Because only one sgRNA and a limited number of plants from limited generations were investigated for off-target mutations, the data cannot rule out the possibility of low-frequency off-target events induced by the same sgRNA and high-frequency off-target events induced by other sgRNA variants (Feng et al. 2014).

Based on the above analysis of the results in the two reports, it is not abnormal that we observed high-frequency off-target mutations in Arabidopsis in our study (Figs. 1, 2), although the sgRNA was predicted to have a high specificity score (Haeussler et al. 2016, Hsu et al. 2013). Actually, the observations in this study were also inconsistent with those in our previous reports, wherein we had suggested that the same sgRNA was specific (Wang et al. 2015 Xing et al. 2014). The main reason for this inconsistency in results is that our previous conclusions were based on only one or two T1 plants, and that different promoters were used to drive Cas9. Consistent with this notion, in our previous report on sgRNA targeting the ABI1 gene, the investigation of eight T1 abi1 mutants and two T2 populations for off-target mutations generated similar results to that observed in the present study (Zhang et al. 2017b). Out of the 8 T1 abi1 mutants, 1 (1/8 or 13%) T1 plant harbored heterozygous or chimeric off-target mutations in AT5G02760 with 2 mismatches, and all the 8 lines harbored no off-target mutations in ABI2 with only 1 mismatch and the other 2 genes with 3 mismatches. Analysis of 52 T2 plants from the T1 line harboring off-target mutations in AT5G02760 showed that 85% of the T2 (44/52) plants harbored off-target mutations in AT5G02760, and 17% (9/52) harbored off-target mutations in ABI2 and both genes. Analysis of 41 T2 plants from the other T1 line harboring no off-target mutations indicated that 10% of the T2 (4/41) plants harbored off-target mutations in AT5G02760, 4.9% (2/41) harbored off-target mutations in ABI2, and 2.4% (1/41) harbored off-target mutations in both genes (Zhang et al. 2017b). Consistent with the results in the present study (Fig. 2), these findings suggest that off-target effects are aggravated in the next generation. It will be interesting to use our egg cell-specific promoter-controlled (EPC) CRISPR/Cas9 system to determine the specificity of GAI-sgRNA1 since its specificity score was 64 relative to the range of 0–100, much lower than that (94) of the sgR-ETC2 (Haeussler et al. 2016, Hsu et al. 2013).

The high-frequency off-target mutations in the CPC gene could be attributed to some sequence features such as position, distribution, and identity of mismatches (Tsai and Joung 2016 Wu et al. 2014). Although the off-target site in the CPC gene has three mismatches with the sgRNA-ETC2, the first mismatch located at the first base distal to PAM is usually tolerated by the CRISPR/Cas9 system (Fig. 1). The second mismatch is also far away from the PAM and/or the two mismatches are situated far from each other, which may also account for the high-frequency off-target mutations in the CPC gene rather than the TRY gene or AT5G50230 (Fig. 1). The PAM-proximal

11-nt were defined as the seed region for Cas9 cutting activity and mismatches in the region are less tolerated (Wu et al. 2014). In some other assays, the seed region was narrowed down to PAM-proximal 5-nt (Wu et al. 2014). Different concentrations of the CRISPR/Cas9 complex and duration of Cas9 binding and cleavage may be responsible for the observed variations in the length of the seed detected by different assays. Therefore, it is not strange for the high-frequency off-target mutations in the CPC off-target site harboring a mismatch in the 8th nt in the PAM-proximal position (Fig. 1). Similar to this study, our previous investigation involving sgRNA-ABI1 indicated that the off-target sites also harbor a mismatch in the 9th nt in the PAM-proximal position (Zhang et al. 2017b). The frequency of off-target events was also affected by mismatch identity and could be largely indicated as: rN:dT ≥ rU:dG >> rC:dC >> rA/rG:dA/dG (Doench et al. 2016 Tsai and Joung 2016 Tycko et al. 2016). The 8th and 9th mismatches mentioned earlier were all of the rC:dT mismatch, suggesting that this mismatch identity was frequently tolerated and contributed to the off-target effects in plants. The observation that the off-target site harboring the two mismatches to the sgRNA targeting ABI1 displayed higher frequency off-target effects than that harboring one mismatch (Zhang et al. 2017b) indicates that more factors should be considered in the development of more precise off-target prediction algorithms that are based on large training data sets from high-throughput experiments.

Because our EPC system is a relatively short-time expression system (Wang et al. 2015), it seemed that it should have a significantly lower off-target frequency than systems driven by constitutive promoters, including 2 × 35S, UBQ1, UBQ10, and PcUbi4-2 (Fauser et al. 2014 Feng et al. 2014 Peterson et al. 2016). On the contrary to this supposition, we observed high-frequency off-target mutations in the CPC gene, which suggests that in the EPC system, a high dosage of the CRISRP/Cas9 complex in egg cells and one cell-stage embryos compensated the short duration for off-target mutations. Consistent with this notion, comparison of the results obtained with tRNA-sgRNA(m) and tRNA-sgRNA(o) suggested that the increased dosage of the complex significantly enhanced the frequency of off-target mutations: 66.7 versus 20.0% (Fig. S1).

It is comprehensible that tRNA-sgRNA(m) generally has a significantly higher editing efficiency than tRNA-sgRNA(o) (Xie et al. 2015) and sgRNA(m) (Dang et al. 2015). However, the present study showed that tRNA-sgRNA(o) had a markedly lower editing efficiency than sgRNA(o), quite contrary to our anticipation (Figs. 3, 4, S1 and S2). This finding may be attributed to three aspects. First, the EPC system might be much more sensitive to fluctuations in effective concentrations or activities of the CRISPR/Cas9 complex than systems driven by constitutive promoters. Consistent with this notion, different terminators (Wang et al. 2015) and even the mCherry cassette behind the terminator (Fig. 3) significantly affected the editing efficiency of the EPC system. In addition, high-specificity SpCas9 variants induced lower efficiency mutations in the T1 plants than their wild-type counterpart, indicating that the EPC system was more sensitive to the fluctuation in the activities of the CRISPR/Cas9 variants. For constitutively expressed CRISPR/Cas9 systems, low levels of sgRNA, if existing, from tRNA-sgRNA(o) could be compensated by the extended duration of expression or activity of the complex, thus leading to an overall high editing efficiency (Xie et al. 2015). Second, the U6 promoters we used might be more sensitive to 4 × T, a potential terminator of Pol-III promoters in the original sgRNA scaffold than the OsU3 promoter. Third, the tRNA secondary structure (the cloverleaf structure) formed after transcription might enhance termination at the 4 × T sites. Whatever the reason, the present study observed that the tRNA-sgRNA(m) was the optimal form that facilitated the successful application of not only the mCherry cassette in counter-selection of Cas9-free plants (Gao et al. 2016), but also high-specificity Cas9 variants (Chen et al. 2017 Kleinstiver et al. 2016 Slaymaker et al. 2016) in avoiding the occurrence of off-target effects.

Our results also suggest that high-specificity SpCas9 mutant variants require much higher concentrations to maintain high editing efficiency than the wild-type counterpart (Fig. 5), although it remains to be determined whether the mutant sgRNA scaffold affected their editing efficiencies. Driven by constitutive and strong promoters in combination with the tRNA-sgRNA(m) fusion strategy, these SpCas9 variants can be used for high-specificity and high-efficiency genome editing in crops. Particularly in geminivirus-mediated CRISPR/Cas9 systems, the editing efficiency of high-specificity SpCas9 will be greatly strengthened since DNA replicons harboring Cas9 and sgRNA cassettes transiently amplify hundreds of copies in a plant cell, thus leading to very high concentrations of the CRISPR/Cas9 complex in a cell (Cermak et al. 2017). Hence, together with the rapid evolution of integrated applications of geminivirus-based replicon systems and CRISPR/Cas9 systems, high-specificity SpCas9 will be particularly useful in avoiding off-target mutations caused by high-dosage CRISPR/Cas9 complexes in a cell (Gil-Humanes et al. 2017 Wang et al. 2017).

One of our unexpected but interesting findings in this report was the high-frequency T-DNA insertions into cleavage sites (Figs. 6, S3–S5). This finding could facilitate the experimental design for targeted integration of transgenes (Li et al. 2016 Salomon and Puchta 1998 Tzfira et al. 2003) and may trigger additional concerns for mutation analysis. First, when encountering a failure in PCR amplification of a target region, T-DNA insertions should be considered as a possibility. Second, in previous reports, the mutation types that were identified as insertions of unknown large fragments might be re-considered as T-DNA insertion events. Third, for T1 Arabidopsis mutants generated using the EPC system or T0 mutants generated from embryogenic callus, ratios of homozygous or biallelic mutants are subject to overestimation and underestimation, respectively. The targeted integrations of two copies of T-DNA into the two alleles of the CPC gene in this report or the HAB1 gene in a previous study might also represent novel knowledge of Agrobacterium-mediated T-DNA insertions into plant genome. First, CRISPR/Cas9-mediated DNA cleavage could be completed before T-DNAs are integrated into the genome of target cells. Second, for Arabidopsis floral dip transformation (Desfeux et al. 2000), random integration of T-DNA usually occurs before fertilization, whereas CRISPR/Cas9-mediated targeted integration of T-DNAs occurs after fertilization.

Since the sgR-ETC2 induced off-target mutations in CPC with high frequency, in TRY with medium frequency, and in AT5G50230 with low frequency under detectable level, our results suggest that carefully selected target sites largely guarantee high specificity. This observation was supported by our previous report, wherein we detected sgR-ABI1-induced off-target mutations in ABI2 and AT5G02760, but did not detect off-target mutations in AT2G25070 and AT3G17090. The intended on-target sites should have no potential off-target sites that harbor less than three mismatches and easily tolerated mismatch features. In addition, awareness is advised when targeting multiple highly homologous genes because off-target sites are possibly adjacent to on-target sites, which may lead to enhanced off-target effects, similar to the case reported in this study. Overall, we recommend using combinatory forms of the following six strategies to avoid off-target effects in plants (Tycko et al. 2016). First, high-specificity targets should be carefully selected using in silico predictions (Haeussler et al. 2016). Second, Cas9-free mutants should be isolated as far as possible (Gao et al. 2016 Lu et al. 2017). Third, when necessary, high-specificity SpCas9 variants in combination with the tRNA-sgRNA(m) fusion method can be used (Chen et al. 2017 Kleinstiver et al. 2016 Kulcsar et al. 2017 Slaymaker et al. 2016 Xie et al. 2015 Zhang et al. 2017a). Fourth, when necessary, SaCas9 or other orthologs with higher specificity can be used (Steinert et al. 2015). Fifth, when necessary, paired nickases can be used (Fauser et al. 2014). Last, when necessary, DNA-free methods can be employed (Liang et al. 2017 Svitashev et al. 2016 Woo et al. 2015).

Collectively, this is the first report on the observations of high-frequency off-target mutagenesis induced by CRISPR/Cas9 in plants our results suggest that in plants harboring CRISPR/Cas9 components, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful to improve genome editing efficiency and specificity in plants and other organisms.


Developing a novel artificial rice germplasm for dinitroaniline herbicide resistance by base editing of OsTubA2

Chemical herbicides, which are commonly used to kill weeds in the field, have been extensively applied and transformed the way of weed management in modern agriculture due to its efficiency, ease of use and relatively low cost. In last decades, a large number of transgenic herbicide-tolerant crops (i.e. corn, soybean, cotton, rice) have been developed and commercialized, reshaping the global seed market (Schutte et al., 2017 ). However, the use of chemical herbicides (i.e. ALS inhibitors, EPSPS inhibitors, ACCase inhibitors) has also drastically increased, resulting in the occurrence of weed species resistant to these herbicides. Thus, developing novel herbicide-resistant crops to diversify weed management is of great value in slowing the evolution of weed resistance to herbicides and to maintain sustainable crop production in the future. Thanks to cutting-edge CRISPR-mediated base editing technologies which have emerged lately (Ren et al., 2019 Wang et al., 2020 Yan et al., 2018 ) important genetic variations related to any herbicide resistance can be manipulated directly and rapidly by the relevant base editors. Tubulin genes have been reported to endow resistance to trifluralin and other dinitroaniline herbicides in a number of crops (Chu et al., 2018 Lyons-Abbott et al., 2010 ). Compared to other major herbicides, the frequency of dinitroaniline resistance in weeds is quite low (Heap, 2014 ). This is likely because mutations in tubulin genes could affect microtubule polymerization, an important process in cell division and elongation, and consequently lead to plant death. Therefore, tubulin genes are promising target genes to develop herbicide-resistant germplasms for future crop breeding. In this study, we successfully generated a novel artificial rice germplasm with trifluralin and pendimethalin resistance without fitness penalty by precisely editing the endogenous OsTubA2 gene within the rice genome. Theoretically, this important trait can also be rapidly introduced into other major crops using CRIPSR-mediated adenine base editors.

It has been previously shown that a Met-268-Thr mutation in the α-tubulin gene EiTUA1 correlates with dinitroaniline resistance in a number of goosegrass (Eleusine indica) biotypes (Yamamoto et al., 1998 ). Proteins belonging to the α-tubulin and β-tubulin family are highly conserved, showing up to 88% amino acid similarity (Rao et al., 2016 ). Therefore, we hypothesized that introduction of this point mutation into the rice genome might transform common rice into a herbicide-tolerant variety. To this end, precise base editing of the α-tubulin homologue gene OsTubA2 (LOC_Os11g14220) was carried out using the previously described rice adenine base editor rBE14 (Yan et al., 2018 ). A sgRNA corresponding to a NGG PAM was designed to target the complementary genomic DNA strand of OsTubA2 at T1981 site (Figure 1a). The oligos were synthesized, constructed and shuttled into rBE14 binary vector through Gateway recombination as described previously (Yan et al., 2018 ). The rBE14/sgRNA system was then introduced into rice cultivar Kitaake (Oryza sativa spp. japonica) through Agrobacterium-mediated transformation.

Rice plants were regenerated from the independent calli and genotyped directly by Sanger sequencing of the target region. Out of 63 independent rice lines obtained, 8 lines (12.7% efficiency) were identified with A > G conversion occurring at T1981, replacing the Met268 residue with threonine residue (Figure 1b). All eight mutant lines were heterozygous and had no random indels detected. The potential off-targets of OsTubA2-targeting rBE14/sgRNA in the rice genome were predicted. No nucleotide changes at the potential off-target sites were detected in the eight positive transgenic lines (Figure 1c).

Next, T1 progenies of each OsTubA2-edited T0 lines were genotyped to determine the lines in which the T-DNA was segregated out. PCR amplification with specific primers corresponding to the Cas9, sgRNA and Hyg transgenes was conducted. As shown in Figure 1d ande, some T1 individuals lacked the transgenes due to genetic segregation. Furthermore, Sanger sequencing revealed that some plants (i.e. #2-5, #2-10 etc.) were homozygous. Given that germination of wild-type Kitaake seeds is sensitive to dinitroaniline herbicide treatment (Figure 1f), a phenotypic and genotypic analysis was further conducted. Rice seeds of T1 population of heterozygous T0 lines #2 and #5 were germinated in cylinders containing 6.6 mg/L pendimethalin which is sufficient to inhibit the hypocotyl and root growth of wild-type rice. All homozygous seeds carrying the M268T mutation showed resistance to pendimethalin treatment, whereas germination of wild-type seed was arrested. On the other hand, heterozygous plants exhibited different resistance phenotypes according to the growth of roots and hypocotyls under the conditions tested, it likely results from the dosage effect of M268T mutation (Figure 1g andh). We also analysed the resistance to 4.0 mg/L trifluralin (another type of dinitroaniline herbicides) which can inhibit the root and hypocotyl growth of wild-type rice. When trifluralin was applied to T1 progenies, similar phenotypes were observed (Figure 1h). Thus, the M268T mutation of OsTubA2 truly confers resistance to both trifluralin and pendimethalin herbicides in rice and is stably inherited in the subsequent generations.

All the OsTubA2-edited plants lacking the T-DNA transgenes were grown under natural light conditions in the greenhouse. The plants were morphologically indistinguishable from the wild-type plants (Figure 1i). Furthermore, the thousand-grain weight and germination rate of T2 seeds were investigated, no significant difference was observed between M268T and wild-type plants (Figure 1j). Together, our results indicate that introduction of Met-268-Thr mutation in OsTubA2 by base editing does not cause growth penalty in rice.

Genetic diversity of OsTubA2 gene in 4,726 re-sequenced rice accessions was investigated using RiceVarMap v2.0 (Zhao et al., 2015 ). The data indicate that M268 in OsTubA2 has not been targeted by natural or human selection during rice domestication (Figure 1k). Thus, M268T plants are a novel artificial germplasm with a great potential for future rice improvement. A number of α-tubulin genes from other major economic crops, including wheat, maize, barley, sorghum, oilseed rape, cotton and soybean, were further analysed. Sequence alignments revealed that the sequence of M268 locus is highly conserved among different species (Figure 1l), opening the possibility that M268T-mediated dinitroaniline resistance can be rapidly introduced into other crops through precise base editing.

In summary, our study shows that the M268T mutation in the endogenous OsTubA2 gene, generated by adenine base editor, endows dinitroaniline herbicide resistance in rice without inducing fitness cost. Any other rice cultivars or cash crops can be enhanced with the herbicide resistance trait using this strategy in the future.



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