Abstract
CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.
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Introduction
Since its first description as programmable nuclease1, the use of Cas9 has revolutionized biology. In plants, CRISPR/Cas systems are now widely used for genome editing in basic science as well as for trait improvement in breeding2,3,4,5. Beside that, CRISPR/Cas can also be used to study DSB repair itself. Multiple studies on DSB repair in unique genomic sequences have been performed but very little is known on how repeats are repaired within repetitive sequences6.
The ribosomal RNA genes (rDNA) of Arabidopsis thaliana are arranged in up to 750 10-kb-long tandem repeats per haploid genome7,8. Each repeat consists of the 18S, 5.8S, and 25S rRNA genes, separated by two conserved internal transcribed spacers (ITS1 and ITS2) and a variable intergenic spacer (IGS).
DSBs can be repaired by two pathways: non-homologous end-joining (NHEJ) or homologous recombination (HR)9. In somatic plant cells, the error-prone NHEJ pathway is the prevalent repair mechanism, which can be subdivided into the KU-heterodimer- and LIG4-dependent classical NHEJ (cNHEJ) pathway and the POLQ-dependent microhomology-mediated end-joining (MMEJ) pathway. Whereas cNHEJ mediates the direct ligation of the broken ends, often resulting in smaller insertions and deletions, MMEJ utilizes microhomologies (MH) close to the break sites, resulting in a loss of sequence between the MH10,11. Repair via HR requires a template and, in the case of tandem repeats, might result in repeat loss. Recently, it was shown that HR is downregulated within the 45S rDNA during meiosis, and that DSBs within this region are repaired via classical NHEJ (cNHEJ)12. However, it has been shown that HR plays a major role in the somatic repeat loss of 45S rDNA in the caf-1 mutant background in Arabidopsis13. To clarify the situation, the initial goal of this study was to directly induce DSBs within the 45S rDNA repeats by Cas9 to define by which pathway they are preferentially repaired. Here, we show that Cas9-mediated DSBs in 45S rDNA are mainly repaired by cNHEJ and furthermore describe a system for the controlled induction of cell death by targeting functional repetitive DNA.
Results
DSB repair in the variable region of the 45S rDNA
To determine the mechanism of DSB repair in the 45S rDNA in somatic cells, we induced DSBs within the 45S rDNA repeats using Staphylococcus aureus Cas9 (SaCas9)14. The target site was selected in the variable IGS region, so that only a subset of repeats was cleaved (Fig. 1a; Supplementary Table 1). As a control, we designed a gRNA targeting the non-essential ADH1 gene (Ubi-SaCas9-ADH1) which induces a unique DSB. Col-0 plants were transformed with the expression vector, Ubi-SaCas9-IGS, as well as the control vector. After two weeks of growth, 40 transgenic plants per line were selected and pooled for extraction of genomic DNA. Subsequently, the 45S rDNA target site was amplified and subjected to next-generation sequencing (NGS) to determine the repair pattern. The deep sequencing analysis revealed that the majority of mutations at the break site were deletions, most of them smaller than 10 bp, and that the repair occurred without MH (Fig. 1b and c). Only a tiny fraction of reads showed insertions or InDels at the break site. These results strongly imply that the cNHEJ is the major DSB repair pathway within the 45S rDNA. To verify this, DSBs were induced in the 45S rDNA of DNA repair mutants at the same target site. The cNHEJ mutants, ku70-1 and lig4-5, as well as the HR mutant, rad54-1, were transformed with the expression and the control construct. The RAD54 mutant was chosen because it is, in contrast to other HR factor mutants, not sterile but has a strong deficiency in somatic HR15. After two weeks of growth, the survival rates of all transformed mutant lines were determined and normalized to Col-0. We expected that an insufficient 45S rDNA repair, due to loss of DNA repair factors, should result in a reduction of the survival rate. Indeed, both cNHEJ mutant lines, ku70-1 and lig4-5, showed a dramatic reduction in survival rate, when the 45S rDNA was cleaved (Fig. 1d). In contrast, no reduction of the survival rate could be observed in the rad54-1 HR mutant line. Thus, cNHEJ plays a key role in 45S rDNA DSB repair in somatic cells.
DSB induction in the conserved region of the 45S rDNA
Next, we used the two intergenic transcribed spacer (ITS) regions as target sites, which are conserved between all repeats. Two gRNAs were designed for ITS1 (Ubi-SaCas9-ITS1-1 and Ubi-SaCas9-ITS1-2) and one guide for ITS2 (Ubi-SaCas9-ITS2-1) (Fig. 1a; Supplementary Table 1). Again, the gRNA targeting the non-essential ADH1 gene (Ubi-SaCas9-ADH1) was used as a control, which showed an editing efficiency of 60% (Supplementary Fig. 1a). Transformation with the T-DNA targeting ADH1 proved to be efficient: 3.2% of the progeny were gentamycin-resistant. In contrast, a dramatic decrease in transformants was observed in the ITS target lines, with a reduction of 96% in case of Ubi-SaCas9-ITS1-1 as well as Ubi-SaCas9-ITS1-2 and a reduction of 99% in case of Ubi-SaCas9-ITS2-1 (Fig. 2a). The fact that quantitative real-time PCR showed no alteration in 45S rDNA copy number in the surviving seedlings suggests that in these few cases, the T-DNA was either truncated or silenced, resulting in insufficient DSB induction (Supplementary Fig. 1b and c). To prove that the nuclease activity of the SaCas9, and not just simple binding of the protein to the rDNA, is required, an additional set of control lines was transformed with catalytically inactive SaCas9 (dCas9) targeting ADH1 (Ubi-dCas9-ADH1) and ITS2 (Ubi-dCas9-ITS2-1). The survival rate determined in both dCas9 approaches was similar and comparable to that of the Ubi-SaCas9-ADH1 control line (Fig. 2b). Thus, the sgRNA ITS2-1 was only able to induce a lethal effect in combination with an active nuclease, demonstrating that massive DSB induction is responsible for the lethal effect. This result was surprising, considering that a recent study reported a dramatic reduction of copy number, but not lethality, when another Cas9 orthologue, namely Streptococcus pyogenes Cas9 (SpCas9), was used for DSB induction16. This difference might have been caused by the fact that SaCas9 is superior in DSB induction in comparison to SpCas9 in Arabidopsis14,17. Our results clearly demonstrate that targeting the conserved part of the 45S rDNA with a highly efficient nuclease is lethal for the transformed cells (Fig. 2c). Based on this observation, we speculated that massive DSB induction could be used as a tool to induce cell death during plant development. Thus, if Cas9 expression was confined to specific growth phases or specific organs, inducing cell death in a highly specific and cell-autonomous manner, a novel form of tissue engineering could be developed (Fig. 2c). By using this system, named CRISPR-Kill, we expected to be able to obtain plants devoid of individual organs or cell types.
CRISPR-Kill for elimination of floral organs
As a proof of concept, we decided to test Cas9 expression under control of the promoter of the floral homeotic gene APETALA1 (AP1, At1g69120). AP1 is a key player for flower meristem identity. It is highly expressed at the early stages of flower development and later gets restricted to specific tissue where it is required for sepal and petal development18 (Fig. 3a). The ap1 mutant shows a disturbed flower architecture where, instead of petals, carpel and stamen are formed (Fig. 3b). We were eager to test if the use of CRISPR-Kill would enable us to specifically eliminate petal tissue. For this approach, the most efficient guide, ITS2-1, was chosen, while the ADH1 guide served as a control. We expected that the utilization of the AP1 promoter would restrict SaCas9 expression exclusively to the early floral meristem and, later on, to sepals and petals. After determination of the transformation rate, no significant reduction could be observed in pAP1-SaCas9-ITS2-1 in comparison to pAP1-SaCas9-ADH1 T1 plants (Fig. 3c). After 4 weeks, 133 independent pAP1-SaCas9-ITS2-1 T1 plants were analyzed for phenotypical deviations in comparison to the control lines. While the control plants showed normal growth, a spectrum of different phenotypes was observed for the CRISPR-Kill plants (Table 1 and Fig. 3d). A very small fraction of CRISPR-Kill T1 lines had died, while one-fifth showed a strong phenotype, characterized by a lack of flowers or lack of sepals and petals. Another fifth showed a weak phenotype, characterized by a full set of sepals, but an incomplete set of petals. In almost half of all lines, an intermediate phenotype could be observed, described by the presence of, at least some, sepals in combination with varying numbers of petals. This intermediate phenotype also consists of the ap1 mutant phenotype (19.8%), characterized by a disturbed floral architecture. To determine whether there is a correlation between the strength of the phenotype and the amount of induced DSBs in the respective line, we isolated floral tissue and performed deep sequencing analysis of the ITS2 target site. In total, two ADH1 control lines (#81 and #82), as well as ten ITS-2-1 lines were analyzed for InDels. The latter ones were subdivided based on their phenotype in ‘no phenotype’ (#5 and #3), ‘intermediate phenotype’ (#1, #4, and #2), and ‘strong phenotype’ (#26 to #30) (Fig. 4). Due to the fact that only a minor portion of respective cell types survived elimination and DNA from adjacent tissues was co-purified, we expected to detect InDels as indications of DSB repair in only a small fraction of reads. The reads of the two control lines showed no InDels at the break site. Occasionally, 1 bp deletions (0.15% for #81 and 0.16% for #82) could be observed, reflecting the natural variances within the repeats. In contrast, all CRISPR-Kill lines that showed a distinguishable phenotype revealed InDels at the break site, with mutation frequencies proportional to the severeness of the phenotype. In the CRISPR-Kill lines with the ‘intermediate phenotype’ mutation, frequencies between 0.31% and 0.99% could be observed. In turn, the CRISPR-Kill lines with the ‘strong phenotype’ showed mutation frequencies between 1.60% and 2.14% (Supplementary Figs. 2 and 3). These findings demonstrate that efficient organ elimination can be achieved by using the CRISPR-Kill system and that the strength of the phenotype is correlated with the efficiency of DSB induction.
Extension of CRISPR-Kill to target centromeric repeats
As the cleavage of 45S rDNA tandem repeats for tissue elimination was highly efficient, we speculated whether the cleavage of other repetitive elements would lead to a comparable result. The 178 bp centromeric sequences represent another highly repetitive cluster of functional tandem repeats19,20. Therefore, the gRNA of the CRISPR-Kill system was programmed to target the conserved 178 bp repeat for DSB induction (Ubi-SaCas9-Cen) (Fig. 5a). Transformation of Col-0 with this construct resulted in a reduction of resistant seedlings by 80% in comparison to the ADH1 control (Fig. 5b). In contrast, AP1-specific expression (pAP1-SaCas9-Cen) did not reduce the number of transformed seedlings (Fig. 5c). The phenotypical analysis revealed the ap1 mutant phenotype as the most abundant, confirming the successful organ elimination (Fig. 5c). Interestingly, in these 40 independent lines the phenotype was much more uniform than in pAP1-SaCas9-ITS2-1.
CRISPR-Kill for elimination of lateral roots
To demonstrate the versatility of the system, we used CRISPR-Kill to modify root development. In A. thaliana, the pericycle cells form lateral roots (Fig. 6a). Thus, elimination of this cell type during root development was expected to hinder lateral root formation. Using the XPP (xylem pole pericycle) promoter, which is exclusively expressed in the pericycle21,22, we transformed Col-0 plants with XPP-SaCas9-ITS2-1, XPP-SaCas9-Cen constructs, and pXPP-SaCas9-ADH1 as a control (Supplementary Fig. 4a). Due to their early expression, a slight reduction of transformation efficiency could be observed in case of the two pXPP-CRISPR-Kill constructs compared to the control (Supplementary Fig. 4b). In all three approaches, 20-day old transgenic T1 plants were checked for lateral root growth. In the 45S rDNA CRISPR-Kill line, a three-fold decrease was observed in lateral root numbers, whereas the centromere CRISPR-Kill line showed a two-fold decrease (Fig. 6b and d). In addition, the relative lateral root length was determined (Supplementary Fig. 4c). Again, a three-fold decrease compared to the control line was observed in the rDNA-targeting line and a two-fold decrease in the centromere-targeting line (Fig. 6c and d). A detailed analysis of individual seedlings showed that, by using CRISPR-Kill, a larger portion of plants hardly developed any lateral roots (Supplementary Fig. 4d and e). This indicates that the severeness of the induced phenotype might vary depending on the T-DNA integration site, as seen in the case of pAP1-SaCas9-ITS2-1 in flower development. As we were interested in whether the phenotypical effects would be reproducible in the following generation, we analyzed root development in presence of XPP-SaCas9-ITS2-1 in the T2 generation. Progeny of T1 plants were selected for presence of the CRISPR-Kill construct and respective plants were analyzed as described above. For both lateral root number and relative lateral root length, again, a 3-fold decrease could be observed compared to the control (Fig. 7 and Supplementary Fig. 5).
Discussion
Taken together, we were able to demonstrate that SaCas9-mediated induction of multiple DSBs in 45S rDNA and centromere repeats can be harnessed to trigger cell death in a cell-autonomous way, resulting in the highly efficient elimination of selected plant cell types. A broad spectrum of phenotypic outcomes was generated, indicating that this system might be a valuable tool to accomplish various stages of cell type or organ elimination. The CRISPR-Kill approach differs massively from others, which conditionally knock out genes in specific cell types to prevent pleiotropic effects or lethality23,24. In addition, it expands the method pool of traditional cell elimination approaches. The most common methods for cell elimination rely on the tissue-specific expression of bacterial toxins, such as the barnase toxin or diphtheria toxin A (DTA). Both systems have been used in plants before to eliminate certain flower tissues, i.e., reproductive organs. In tobacco and Arabidopsis, the barnase system could be applied to induce female sterility, by targeting the stigmatic secretory zone of pistils, or male sterility, by targeting the mature stamen, respectively25,26. Similarly, using DTA in combination with the promoter of the floral homeotic protein AP3, expressed in petal and stamen tissue, the respective tissue could be eliminated and, thus, male sterility could be induced27. Next to toxins, also the UV laser ablation technique has been used successfully for cell elimination, i.e., in root endodermis cells to analyze wound response and regeneration processes28,29.
The major advantage of the CRISPR-Kill system is the broad range of applications. Besides tissue elimination, it can be applied for purposes not aiming for cell elimination but rather genome engineering. In contrast to the classical methods that eliminate cells in a rampant manner, the CRISPR-Kill system targets specific genomic sites and, thus, can be programmed to cleave individual chromosomes. So far, it has only been shown in mammals that individual chromosomes can be eliminated through the induction of multiple DSBs30,31. Transferring this to plants, CRISPR-Kill-based chromosome elimination might be an attractive application in plant hybrids, especially if coupled with chromosome restructuring32,33.
The CRISPR-Kill system is easy to adapt for use in other plant species, or even other multicellular eukaryotes, such as animals or fungi, due to the high conservation of the target regions. It will be interesting to combine it with an inducible and two-component system to control Cas9 expression, so that CRISPR-Kill can be controlled both temporally and spatially22,34.
CRISPR-Kill could also be an attractive tool for applications in synthetic biology. Cell-type specificity is a common characteristic of multiple biochemical pathways in plants, especially in secondary metabolism. Metabolites important for plant defense are often produced in specific cell types to avoid the peril of autotoxicity and to maximize the defense function35. Using the CRISPR-Kill system, the change of secondary metabolite composition within a plant and also the elimination or even production of new specific metabolites might become achievable. Thus, food production as well as pharmaceutical applications could benefit from this technology.
Methods
T-DNA constructs
DNA constructs were generated using the Gateway®-compatible (Thermo Fisher Scientific Inc., https://www.thermofisher.com) pDe-Sa-Cas9 and pEn-Sa-Chimera plasmids14. The kanamycin resistance cassette was exchanged with gentamycin using the restriction enzymes PmeI and SbfI. The target sequences for break induction were ligated with pEn-Sa-Chimera as oligonucleotides, using AS1-AS12 (see Supplementary Table 1 for oligonucleotide sequences). Via Gateway LR reaction, the sgRNA expression cassette was transferred into the pDe-Sa-Cas9 vector, resulting in Ubi-SaCas9-ADH1, Ubi-SaCas9-IGS, Ubi-SaCas9-ITS1-1, Ubi-SaCas9-ITS1-2, Ubi-SaCas9-ITS2-1, and Ubi-SaCas9-Cen. The AP1 promoter was amplified from Arabidopsis genomic DNA using the primer combination AS13 and AS14 (see Supplementary Table 2 for oligonucleotide sequences). The XPP promoter was synthesized (BioCat GmbH, https://www.biocat.com) and later amplified using the Primer combination AS15 and AS16 (see Supplementary Table 2 for oligonucleotide sequences). By EcoRI restriction enzyme digestion and Gibson Assembly® (New England Biolabs Inc., https://international.neb.com), the PcUbi4-2 of Ubi-SaCas9-ADH1 and Ubi-SaCas9-ITS2-1 and Ubi-SaCas9-Cen were exchanged with the AP1 and XPP promoter to generate the tissue-specific constructs. For alignment and analysis of Sanger sequencing data, the software ApE was used. All constructs are available on request.
Plant material and growth conditions
Experiments were performed in the Arabidopsis thaliana background Columbia-0 (Col-0). In addition, the T-DNA insertion lines ku70-1 (At1g16970, SALK_123114)36, lig4-5 (At5g57160, SALK_095962)37, and rad54-1 (At3g19210, SALK_038057)15 were obtained from the SALK collection38. Plants were cultivated in the greenhouse on soil (1:1 mixture of Floraton [Floragard] and vermiculite [2–3 mm, Deutsche Vermiculite Dämmstoff]) with 16 h light (Phillips, Master, TL-D36W/840) and 8 h darkness at 22 °C. When sterile conditions were required, plants were cultured in a CLU-36L4 plant culture chamber (Percival Scientific) with stable conditions of 16 h light at 22 °C and 8 h dark at 20 °C on germination media (GM: 4.9 g/L Murashige and Skoog medium [Duchefa], 10 g/L Suc, and 7.6 g/L; pH 5.7 with potassium hydroxide). Seeds were surface-sterilized using 4% sodium hypochlorite and stratified overnight at 4 °C. For Agrobacterium-mediated plant transformation, floral-dipping was performed.
Deep sequencing
For next-generation sequencing analyses (NGS) of the ADH1 locus, the genomic DNA of a pool of 40 primary transformants was extracted. To analyze repair patterns and SaCas9 efficiency, primers with 6 bp overhangs (AS17-AS20, see Supplementary Table 2 for oligonucleotide sequences) were designed to amplify a fragment containing the target site. For NGS analysis of the 45S rDNA ITS locus, DNA extraction of only the floral tissue was performed. Primer with 32 and 33 bp overhangs was designed (AS29 and AS30) for amplification of the target site. The 300–500 bp amplicons were generated using Q5 High-Fidelity DNA polymerase (New England Biolabs) and later purified using the peqGOLD Cycle Pure Kit (Peqlab). The amplicons were sequenced with the Illumina HiSeq platform at GATC Biotech. The quality of the reads was verified using the CLC Genomics Workbench (Qiagen Bioinformatics) and later analyzed using the CRISPR RGEN TOOL (Cas analyzer39) (http://www.rgenome.net/cas-analyzer/#!). Parameters were set for minimum frequency (n) = 1 and comparison range R = 200, no wild-type marker (r). Afterward, the data were analyzed using R Studio and Excel.
Determination of T1 survival rate
T1 seeds were sown on GM containing the respective selection marker. After 14 days, the number of transgenic plants was counted and put in relation to the total seed number to determine the survival rate.
Quantitative real-time PCR
To determine the amount of 45S rDNA copy numbers, real-time PCR analysis was conducted. Genomic DNA was extracted from 14-day-old A. thaliana seedlings and 0.5 ng of DNA were combined with 5 μL 2x qPCR Master Mix KAPA SYBR FAST (Sigma-Aldrich Chemie GmbH), 0.5 μL of forward and reverse primer (10 mM, AS21-AS28, see Supplementary Table 2 for oligonucleotide sequences) and filled up with distilled water to a total volume of 10 μL. The Light Cycler480 instrument (384-well block system; Roche Diagnostics) was used for analysis. All rDNA genes (18S, 5.8S, and 25S) as well as the reference gene Ubiquitin 10 were tested in three technical, as well as three biological replicates.
Phenotypic analysis
To analyze phenotypical differences in the floral tissue, plants were grown in the greenhouse for 4 weeks. In total, 19 control plants, 133 pAP1-SaCas9-ITS2-1, and 40 pAP1-SaCas9-Cen lines were screened for phenotypical differences and sorted into different categories, dependening on the extent of these differences. Analysis was performed using a binocular microscope and pictures were taken with VisiCam 5 plus using the software IS Visicam Image analyser (VWR, https://www.vwr.com). To analyze phenotypical differences in the root, plants were first selected for transgene integration on selection medium containing PPT for 13 days. In total, 108 control plants of each line were transferred from selection medium to germination medium. After another 7 days, the number of lateral roots was determined. In addition, relative lateral root length, in relation to the main root length, was determined by the Image J add-on SmartRoot40. To analyze the effects of CRISPR-Kill in roots in the T2 generation, T1 lines were propagated to T2 and selected for heterozygous T-DNA integration on selection medium containing PPT. In total, three plants each out of 30 T2 lines were analyzed as described above.
Statistical methods
Differences in survival rates were determined by applying a two-sided, two-sample t-test with unequal variance. For root number and relative lateral root length, a one-way ANOVA was performed: ns P > 0.05, *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
Deep sequencing data that support the findings of this study have been deposited in SRA [accession: PRJNA726366]. Source data are provided with this paper.
Code availability
R code for detailed analysis of NGS data has been deposited to GitHub [https://github.com/PuchtaLab/CRISPR-Kill/blob/main/R-code].
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Acknowledgements
We thank Jessica Sieber and Sakia Gabsi for technical assistance performing experiments, Traudel Wehrle for assisting with cultivation of the plants in the greenhouse, Patrick Schindele and Michelle Rönspies for critical reading of the manuscript, Tobias Zundel for providing an R-based script for bioinformatical analysis of NGS data. This research was funded by European Research Council ERC [Grant number ERC-2016-AdG_741306 CRISBREED] to H.P.
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H.P. and A.S. conceived the research; A.S., F.G., and S.R. designed the research; A.S., F.G., and C.S. conducted the research; A.S., A.D., and H.P. wrote the paper.
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Schindele, A., Gehrke, F., Schmidt, C. et al. Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats. Nat Commun 13, 1502 (2022). https://doi.org/10.1038/s41467-022-29130-w
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DOI: https://doi.org/10.1038/s41467-022-29130-w
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