Applications of CRISPR/Cas9 in agriculture, nutrition, health and disease

Keywords: CRISPR/Cas9, agriculture, nutrition

Abstract

New advances in molecular biology and gene editing technologies that allow for the targeted manipulation of genes in living organisms have a wide range of applications in biomedicine, healthcare, and agriculture. Such approaches can be used to improve the nutritional status of different crops, in order to provide needed micronutrients to vulnerable populations or improve animal feed. One of the most successful genome-editing technologies in recent times, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, has attracted a lot of attention due to its simplicity, efficiency, and versatility. This method is being applied in almost all biological research areas and has a wide range of applications. This review focuses on the applications of the CRISPR/Cas9 system in agriculture, nutrition, health, and disease.

Introduction

Genetics, nutrition, environmental factors, and lifestyle habits all influence an individual’s health. Diet plays a critical role in overall health, as the food we eat and the nutrients they provide influence growth, development, functional abilities and protect against disease. Poor nutrition in both the short- and long-term, can induce stress and increase the risk of developing various diseases. Researchers have utilized different genetic techniques to increase crop quality and nutritional quantity, in an effort to enable farmers to meet the requirements of the increasing population. In recent years, the CRISPR/Cas9 technique has been successfully used by many researchers and agricultural companies to address this issue.

The CRISPR/Cas9 system depends on a single Cas9 protein, which is a DNA nuclease, and a single guide RNA (sgRNA) molecule containing 20 nucleotides. The sgRNA contains nucleotides complementary to the genomic target sequence. Binding of the sgRNA to the target DNA forms an RNA-DNA hybrid that in turn recruits the Cas9 nuclease, which generates a single stranded break (SSB) or a double stranded break (DSB) in the DNA adjacent to the protospacer adjacent motif (PAM), a short region immediately downstream of the target sequence. The Cas9 protein possesses two nuclease domains, namely RuvC and HNH. The RuvC and HNH domains bind to each strand of DNA to generate a blunt-ended DSB. Once SSBs or DSBs are generated, they will be repaired by the cell’s own DNA repair machinery through either non-homologous end joining (NHEJ) or homologous directed repair (HDR) mechanisms in presence of a donor DNA sequence. Mutating both RuvC and HNH domain sequences generate a catalytically inactivated Cas9 protein, which cannot cleave the DNA and is thus named «dead Cas9» (1 – 4). More detailed information on this method and its development have been recently reviewed (5 – 7).

The CRISPR/Cas9 system was initially discovered in bacteria, such as Streptococcus pyogenes, where it protects against infections from DNA viruses or bacteriophages (3, 7, 8). When bacteria are infected with virus, the viral DNA is cut into several fragments and subsequently integrated into the bacterial genome. These viral DNA fragments are stored at a specific locus termed the CRISPR locus in the bacterial genome. The integrated viral DNA fragments at the CRISPR locus are transcribed as RNA (crRNA), while the Cas9 protein is produced from the Cas locus. In the case of a re-infection by the same virus, both the crRNAs and the Cas9 will be present in the bacterial cell. Upon the binding of crRNA to the complementary target DNA released from the virus, the Cas9 protein binds and generates DSBs in the viral DNA. Cas9 cleaves the viral DNA at each crRNA bound, and thus protects the bacteria (Fig. 1). This manner of «remembering» and destroying DNA from similar invaders has now been re-engineered by researchers to enable its use in human, animal cells, plants, and in a wide range of systems/organisms.

Fig. 1 Bacterial CRISPR/Cas defense can be divided into 3 stages. The first stage (integration): The viral DNA is inserted into the CRISPR locus. The second stage (transcription/expression): crRNAs are transcribed from the CRISPR locus and Cas proteins are expressed from the Cas locus. Third stage (interference): In the case of future infections, crRNA and the Cas protein complexes bind and cleave the viral DNA protecting against the infections.

Applications of CRISPR/Cas9

The CRISPR/Cas9 system is turning out to be a groundbreaking innovation and has been successfully used in distinct branches of biology including agriculture and plant genome engineering (Fig. 2).

Fig. 2 Applications of CRISPR/Cas9: CRISPR/Cas9 is currently used in genetic engineering, agriculture, and nutritional research.

CRISPR/Cas9 as a tool in agriculture

Researchers are using CRISPR/Cas9 in several agricultural plant species, by targeting various genes of interest for improved nutrition, enhanced disease resistance and improved tolerance against drought. Here we summarize some of the successful studies where the CRISPR/Cas9 system is applied to modify several agricultural plants.

Wheat plants are sensitive to powdery mildew, due to the mlo gene present in its genome. Researchers utilized CRISPR/Cas9 to knock out mlo alleles, giving heritable resistance to powdery mildew (9). In rice plants, herbicide tolerance and bacterial infections are long-standing problems. Recently, CRISPR/Cas9 has been used to knock-out herbicide tolerance genes, create hybrids (10), and alter susceptibility to bacterial infections (11). The technology has been used to edit recessive mutations in tomato plants preventing needle-like leaves (12). The maize genome has also been successfully modified using the CRISPR/Cas9 technology (13 – 16). Traits that have been altered using this technology include male sterility, lignin biosynthesis, herbicide tolerance, RNA metabolism, secondary metabolism, grain composition, and drought tolerance. Work is also being done to lower the amount of phytic acid (PA) in maize (21). PA is poorly digested in humans and poses a threat to the environment (21). The PA content of maize seeds was reduced by designing two sgRNAs targeting the ZmIPK (Inositol Phosphate Kinase) gene that catalyzes a key step in the PA biosynthetic pathway. The technique has also been used to introduce targeted gene edits in the soybean genome (17), to alter traits such as seed oil, protein composition and herbicide tolerance. In addition to the above, a plethora of data are available where different plants have been edited using CRISPR/Cas9, including Arabidopsis (18), tobacco (18) rice (19), wheat (9), sorghum (20), maize (21), tomato (12), and sweet orange (22). Currently, researchers are also aiming at producing non-browning apples, mushrooms, and potatoes by mutating polyphenol oxidase (PPO) genes (23).

Use of the CRISPR/Cas9 system in metabolic engineering

Applications of CRISPR/Cas9 include extensive research in the field of metabolic engineering, where plant cells are targeted for production of specific metabolites. Alagoz, et al., manipulated the biosynthesis of benzylisoquinoline alkaloids (BIAs) in Papaver somniferum by knocking out the 30-O-methyltransferase (30OMT2) gene via the NHEJ DNA repair mechanism. 40-O-methyltransferase (40OMT2) is a regulatory gene involved in the biosynthesis of codeine, noscapine, papaverine, and morphine via different BIA pathways (24). These manipulations can thus convert valuable medicinal plants into biofactories for the mass production of specific metabolites, simply by introducing breaks in related gene sequences.

Li, et al., targeted the diterpene synthase gene (SmCPS1), involved in tanshinone biosynthesis in Salvia miltiorrhiza, a Chinese herb well known for its vasorelaxation and antiarrhythmic effects (25). Diterpene synthase is an entry enzyme which uses geranylgeranyl diphosphate (GGPP) as its substrate for generating tanshinones. GGPP also acts as a precursor for taxol biosynthesis, thus knocking out SmCPS1 blocks the metabolic flux of GGPP to tanshinone, switching GGPP to taxol synthesis. Agrobacterium rhizogenes mediated transformation using CRISPR/Cas9 generated three homozygous and eight chimeric mutants from 26 independent transgenic hairy root lines of Salvia. Metabolomic analyses revealed zero tanshinone accumulation in the homozygous mutants, and a decreased percentage in chimeric mutants, underlining the efficiency of gene editing with CRISPR/Cas9 (25).

Utilization of the CRISPR-Cas9 system in bacterial cultures

CRISPR-based technology may provide high-resolution genotyping of bacteria (26) since various strains of bacteria harbor different spacers, due to the spacer acquisition’s polarized nature (27). This method has currently proved successful in efficiently genotyping foodborne pathogens, such as Salmonella (27) and E. coli (28). Common problems when using starter cultures in food production are phage infections, antibiotic resistance, and contamination. CRISPR/Cas9-based technology may be used to confer phage resistance in bacteria, by introducing specific sgRNA sequences against different phages (26). One sequence may also confer resistance towards multiple phage strains if they harbor a conserved functional sequence (29). In addition, it is possible to utilize the CRISPR/Cas9 system in a heterologous manner in order to vaccinate bacteria lacking the CRISPR/Cas9-system. Introduction of the system may thus render the bacteria resistant to uptake of any unwanted genetic content (1). Moreover, genes encoding antibiotic resistance, either gained by genetic uptake or spontaneous mutations, can be targeted, resulting in bacterial sensitivity to antibiotics (30, 31). One can also deplete starter cultures of various microbes by introducing self-targeting CRISPR/Cas9 systems, thus depleting the culture of populations in a sequence-specific manner (32). Such microbes may also be neutralized by targeting genes conferring virulence and pathogenicity (31).

CRISPR/Cas9 as a tool for wine yeasts with decreased urea production

The EC1118 and AWRI796 are two commercially available starter yeast strains of Saccharomyces cerevisiae commonly used in wine production. These yeast strains contain the CAN1 arginine permease gene, which increases the presence of urea in the wine. EC1118 and AWRI796 have been genetically modified using CRISPR/Cas9 to reduce the production of urea (33). Elimination of the CAN1 arginine permease has resulted in greatly reduced urea production in both strains (33). When grape musts from Chardonnay and Cabernet Sauvignoncultivars were used as a wine-model environment, the recombinant strains failed to produce urea because of impaired arginine metabolism. In wine production, urea may act as a precursor of ethyl carbamate (34), which has been classified as a carcinogen by the International Agency for Research on Cancer (IARC) (35). This can occur spontaneously, as urea and ethanol react to form ethyl carbamate (33). Thus, CRISPR/Cas9 may serve as a tool to decrease the formation of ethyl carbamate in wine production.

The implication in correcting mutation for hereditary tyrosinemia in rats

In the following section, we summarize some studies where CRISPR/Cas9 was used to correct mutations responsible for metabolic genetic disorders in rodent models.

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutations in fumarylacetoacetate hydrolase (FAH). Due to the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. Shao, et al. have generated a Fah mutant rat model to investigate whether genome-editing can efficiently correct the Fah gene (36). After receiving Cas9-mediated gene correction therapy, HTI rats steadily gained weight and survived. Fah-expressing hepatocytes occupied more than 95 % of the liver tissue nine months after treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. This indicates that CRISPR/Cas9 is a valuable and safe gene therapy strategy for this genetic disease.

CRISPR-Cas9: Biomedical advancements and opportunities

Many studies have applied the CRISPR/Cas9 method in both in vitro and in vivo models to explore diverse cellular pathways, and to make advances in the biomedical research field. Genome editing enables the rapid generation of cellular and animal models, useful in many areas of biological research. Disease mutations are studied by using models, or by making a phenocopy of a particular disorder (37). Genome-wide association studies have found several regions in the genome that harbor potential risks for polygenic diseases such as diabetes, Alzheimer’s, schizophrenia, and autism. Cas9-based multiplex genome engineering holds promise in assessing the roles of these loci, both individually and simultaneously. Effects of genome modifications can be tracked by genome editing stem cells, followed by their differentiation into the cell type of interest (38).

Cas9 mouse lines, which express Cas9 in a constitutive or tissue-specific manner, have been generated by crossing a Cre-depended Cas9 mouse with specific Cre-driver strains. Delivery of specific sgRNAs to the Cas9 mice enabled both ex vivo and in vivo genome editing of neurons, immune cells, and endothelial cells. Simultaneous modeling of lung adenocarcinoma through multiplexing has also been demonstrated (39). Genome engineering holds great promise for regenerative medicine-based therapeutics. Direct genome editing in tissues can be a primary route for treatment. Several proofs of concept studies have proposed such methods for correcting monogenic recessive genetic disorders such as hemophilia (40), cystic fibrosis (41), Duchenne muscular dystrophy (42), tyrosinemia (43), Fanconi anemia (44), and sickle cell anemia (45). Inactivation of a mutant allele by genome editing has been proposed for correcting dominant negative genetic disorders, such as retinitis pigmentosum and transthyretin-related hereditary amyloidosis (38).

Variations in the non-genic (enhancer) regions have been shown to underlie autoimmune diseases. Thus, making DNA manipulations through genome editing a promising therapeutic intervention for their mitigation (46). Besides repairing disorder-associated genes, genome editing-based regenerative medicine can also be used to protect individuals from disease risk by disrupting certain genes. Proof of concept studies in mice has shown that disrupting the Pcsk9 gene in vivo (liver) has therapeutic promise against cardiovascular disease in humans (47). Engrafting Cas9 modified CCR5 human hematopoietic stem and progenitor cells is a promising approach in combating AIDS (48).

The CRISPR/Cas9 system can be used to modulate the virulence of bacterial populations. Phages and conjugative plasmids enabled the delivery of SSNs to microbial populations, using Cas9 programmed to target specific sequences underlying antibiotic resistance and virulence in bacteria (49). Malarial parasite Plasmodium falciparum has been notoriously resistant to efforts of the research community to elucidate its intraerythrocytic developmental genetics, slowing down the development of novel drugs and vaccines. The CRISPR/Cas9 technology has emerged as a fast and efficient tool that has been successfully applied to manipulate or knockout malaria genes (50); a process that used to take an extensive amount of time.

In addition to the above studies, the CRISPR/Cas9 technology has been applied in haematological, neurological, non-cancerous, cancerous and monogenic diseases in various mouse, rat, pig and monkey model systems as well as in humans. This has been excellently reviewed in Pandey, et al., 2017 (51).

Future perspective

The CRISPR/Cas9 technology has witnessed a greatly accelerated development from its role in bacterial immunity to therapeutic use. Prior to the clinical translation of Cas9, its safety and physiological effects still need to be thoroughly assessed and characterized. Concerned authorities must determine whether CRISPR/Cas9 based genome engineering technology applied to crops merits an overall exemption from the regulatory process or a less rigorous regulation. The CRISPR/Cas9 system has a unique ability to change all the three molecules of life, DNA, RNA, and protein, in a customizable manner. It provides scientists with an incredibly powerful tool to improve nutrition, health, and diseases at an unprecedented pace through genetic manipulations that were not thought possible before its discovery.

Conflict of interest: The authors declare no conflict of interest.

References

  1. Sapranauskas R, Gasiunas G, Fremaux C, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011;39(21):9275 – 82.

  2. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109(39):E2579 – 86.

  3. Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816 – 21.

  4. Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173 – 83.

  5. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.

  6. Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827 – 32.

  7. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823 – 6.

  8. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819 – 23.

  9. Wang Y, Cheng X, Shan Q, et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol. 2014;32(9):947 – 51.

  10. Xu RF, Li H, Qin RY, et al. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice. 2014;7.

  11. Jiang WZ, Zhou HB, Bi HH, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research. 2013;41(20).

  12. Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System. Plant Physiology. 2014;166(3):1292 – 7.

  13. Svitashev S, Young JK, Schwartz C, et al. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA. Plant Physiol. 2015;169(2):931 – 45.

  14. Svitashev S, Schwartz C, Lenderts B, et al. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun. 2016;7:13274.

  15. Shi J, Gao H, Wang H, et al. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol J. 2017;15(2):207 – 16.

  16. Char SN, Neelakandan AK, Nahampun H, et al. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnol J. 2017;15(2):257 – 68.

  17. Li Z, Liu ZB, Xing A, et al. Cas9-Guide RNA Directed Genome Editing in Soybean. Plant Physiol. 2015;169(2):960 – 70.

  18. Li JF, Norville JE, Aach J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688 – 91.

  19. Shan Q, Wang Y, Li J, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31(8):686 – 8.

  20. Jiang W, Zhou H, Bi H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013;41(20):e188.

  21. Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics. 2014;41(2):63 – 8.

  22. Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 2014;9(4):e93806.

  23. Nishitani C, Hirai N, Komori S, et al. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system. Sci Rep. 2016;6:31481.

  24. Alagoz Y, Gurkok T, Zhang B, et al. Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology. Sci Rep. 2016;6:30910.

  25. Li B, Cui G, Shen G, et al. Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza. Sci Rep. 2017;7:43320.

  26. Barrangou R, Horvath P. CRISPR: new horizons in phage resistance and strain identification. Annu Rev Food Sci Technol. 2012;3:143 – 62.

  27. Shariat N, DiMarzio MJ, Yin S, et al. The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis. Food Microbiol. 2013;34(1):164 – 73.

  28. Toro M, Cao G, Ju W, et al. Association of clustered regularly interspaced short palindromic repeat (CRISPR) elements with specific serotypes and virulence potential of shiga toxin-producing Escherichia coli. Appl Environ Microbiol. 2014;80(4):1411 – 20.

  29. Barrangou R, Coute-Monvoisin AC, Stahl B, et al. Genomic impact of CRISPR immunization against bacteriophages. Biochem Soc Trans. 2013;41(6):1383 – 91.

  30. Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468(7320):67 – 71.

  31. Selle K, Barrangou R. CRISPR-Based Technologies and the Future of Food Science. J Food Sci. 2015;80(11):R2367 – 72.

  32. Beisel CL, Gomaa AA, Barrangou R. A CRISPR design for next-generation antimicrobials. Genome Biol. 2014;15(11):516.

  33. Vigentini I, Gebbia M, Belotti A, Foschino R, et al. CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production. Front Microbiol. 2017;8:2194.

  34. Weber JV, Sharypov VI. Ethyl carbamate in foods and beverages: a review. Environ Chem Lett. 2009;7(3):233 – 47.

  35. Lee KG. Analysis and risk assessment of ethyl carbamate in various fermented foods. Eur Food Res Technol. 2013;236(5):891 – 8.

  36. Shao Y, Wang L, Guo N, et al. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats. J Biol Chem. 2018;293(18):6883 – 92.

  37. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32(4):347 – 55.

  38. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262 – 78.

  39. Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159(2):440 – 55.

  40. Li H, Haurigot V, Doyon Y, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature. 2011;475(7355):217 – 21.

  41. Schwank G, Koo BK, Sasselli V, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 2013;13(6):653 – 8.

  42. Ousterout DG, Perez-Pinera P, Thakore PI, et al. Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol Ther. 2013;21(9):1718 – 26.

  43. Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol. 2014;32(6):551 – 3.

  44. Osborn MJ, Gabriel R, Webber BR, et al. Fanconi anemia gene editing by the CRISPR/Cas9 system. Hum Gene Ther. 2015;26(2):114 – 26.

  45. Sun N, Zhao H. Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs. Biotechnol Bioeng. 2014;111(5):1048 – 53.

  46. Farh KK, Marson A, Zhu J, et al. Genetic and epigenetic fine mapping of causal autoimmune disease variants. Nature. 2015;518(7539):337 – 43.

  47. Ding Q, Strong A, Patel KM, et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res. 2014;115(5):488 – 92.

  48. Mandal PK, Ferreira LM, Collins R, et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell. 2014;15(5):643 – 52.

  49. Citorik RJ, Mimee M, Lu TK. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol. 2014;32(11):1141 – 5.

  50. Ghorbal M, Gorman M, Macpherson CR, Martins RM, et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol. 2014;32(8):819 – 21.

  51. Pandey VK, Tripathi A, Bhushan R, Ali A, et al. Application of CRISPR/Cas9 genome editing in genetic disorders: A systematic review up to date. J Genet Syndr Gene Ther. 2017; 8:2: