Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Abstract Background Bitter taste receptors (Tas2rs) are generally considered to sense various bitter compounds to escape the intake of toxic substances. Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo. Methods To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues, multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene‐editing technique. A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes. Then, T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds. Perception to taste substance was also studied using two‐bottle preference tests. Results We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique. Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice. But qRT‐PCR results revealed the changed expression profile of mTas2rs gene in taste buds of these mutant mice. With two‐bottle preference tests, these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105. In addition, these mutant mice showed a loss of taste perception to quinine dihydrochloride, denatonium benzoate, and cucurbitacin B (CuB). Gnat3‐mediated taste receptor and its signal pathway contribute to CuB perception. Conclusions These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound–induced responses mediated by these TAS2Rs in many extraoral tissues.


| INTRODUC TI ON
3][4][5] Several studies with Tas2r143-CreERT2 transgenic mouse, 6 Tas2r105-Cre/GFP transgenic mouse, [7][8][9] and Tas2r131-Cre knock-in mouse 10,11 showed that bitter taste receptors were expressed outside the gustatory system.Such solitary chemosensory cells are capable of sensing bacterial quorum sensing molecules in the respiratory tract [12][13][14] and can induce type 2 immune responses to helminth infections in the murine intestinal tract. 4Furthermore, susceptibility to upper respiratory tract infection is associated with TAS2R38 polymorphisms in humans. 15,16Taste receptor polymorphisms are even linked to male infertility by altering sperm cilia motility and downregulating meiotic cell division. 3This indicates that bitter taste receptors have important physiological functions in extraoral tissues in vivo.
8][19] Muroid cluster I contains Tas2r107, Tas2r106, Tas2r105, Tas2r114, and Tas2r104, which have a common ancestor with human TAS2R10. 17,18Tas2r104, Tas2r105, and Tas2r114 are organized as a head-to-tail array within a single 6-kb DNA fragment.This may be regarded as a representative of a single human gene being orthologous to multiple mouse genes, that is, one-to-multiple orthology. 20[23][24] In particular, Tas2r114 with the lowest expression in lingual papillae 25 showed moderate to high expression in kidney, 21 gut, 21 and testis. 24terologous expression analysis of Tas2rs in HEK293T cells showed that these Tas2rs were responsive to different bitter compounds. 25erefore, it is tempting to speculate that these Tas2rs in muroid cluster I may discern a different set of the bitter compounds and have different physiological functions in vivo.To investigate the physiological functions of bitter taste receptor cluster Tas2r106/Tas2r104/ Tas2r105/Tas2r114 in gustatory and extraoral tissues, multiple Tas2rs knockout (KO) mice were produced using CRISPR/Cas9 gene-editing technique.Using two-bottle preference tests, we observed that these Tas2rs mutant mice have a decreased response to the cycloheximide (CYX), quinine dihydrochloride (QHCl), denatonium benzoate (DB), and cucurbitacin B (CuB).

| Animals
This study was approved by the Animal Care and Use Committees of Shanghai Public Health Clinical Center (GW2018-A055-01, 2023-A030-01).Animals were kept and reproduced in a specific pathogenfree animal facility at the Department of Laboratory Animal Science, Shanghai Public Health Clinical Center (five mice per cage), with free access to food and water.

| sgRNA and Cas9 mRNA in vitro transcription
The MIT CRISPR Design Tool was employed to analyze target sites at the Tas2rs gene and Gnat3. 26gRNAs with the highest scores were chosen.A ligation-free PCR approach for sgRNA template synthesis was used in this paper. 27A 60-bp target-specific oligo, including the T7 promoter, was synthesized, and the sgRNA template was generated using PCR with several sets of universal primers.The primers used are presented in Table 1.In vitro transcription and RNA purification were performed using the conventional methods, as previously described. 27nal products were stored at −80°C until further analysis.

| T7EN1 cleavage and sequencing
Mouse genomic DNA was extracted from mouse tail tips using routine methods, as described previously. 28Hieff Canace high-fidelity DNA polymerase (YEASEN, 10135ES80) was used to amplify target sites.Primers are presented in Table 1.Then, T7 endonuclease I investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

K E Y W O R D S
bitter taste receptor, CRISPR/Cas9, genetic mutation, two-bottle preference test, type 2 taste receptors (Tas2rs) TA B L E 1 Oligonucleotides for genotyping, surveyor assay and IVT-1 in this study.(T7EI, M0302L, NEB) was used to digest hybridized PCR products, as described previously. 28Target region amplicons from mutant founders were cloned into a pESI-Blunt vector (YEASEN, 10909ES20) and sequenced by Sangon Biotech.Positive F0 founders were crossed with C57BL/6J mice to screen whether mutation was transferred to the next generation.

| RNA extraction of circumvallate taste buds, reverse-transcription PCR, and quantitative real-time PCR
The procedure to prepare the circumvallate papillae closely followed published protocols. 29,30Briefly, the tongue was immediately removed and placed in ice-cold Tyrode's solution after mice were killed.An enzyme cocktail (1 mg of collagenase A [Roche Diagnostics, Monza, Italy], 2.5 mg of dispase II [Roche Diagnostics], and 1 mg of trypsin inhibitor type I-S [Sigma] in 1 mL of Tyrode's solution) was then injected between the lingual epithelium and the muscle layers.
After incubation in divalent-free Tyrode's solution at room temperature (20°-22°) for 30-40 min with air bubbling, the lingual epithelium was carefully peeled free from the underlying tissue.Finally, circumvallate papillae were softly dissected under a dissecting microscope and immersed in ice-cold Tri-Reagent (Trans, Beijing).Adjacent epithelium without fungiform taste buds was used as control.
Total RNA was extracted from taste buds using Tri-Reagent (Trans) following the manufacturer's protocol.DNAaseI (Roche) was used to eliminate the residual genome; 1 μg of isolated RNAs from each sample was reverse-transcribed into complementary DNA (cDNA) using the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen Biotech).
Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to investigate the expression levels of Tas2rs using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotech) following the manufacturer's protocols; 10 ng of cDNA was added in each tube.The cycling conditions for qRT-PCR were 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s.2][33] All the primers for mouse Tas2rs, Gnat3 are presented in Table 1 and have been used in our previous study. 7,9

| Two-bottle preference tests
The two-bottle preference test was performed as described previously. 37Taste preferences were studied using a 48-h two-bottle preference test with the following taste compounds: sucrose

| Statistical analyses
All data are presented as mean ± standard error.We analyzed the differences among experimental groups using SPSS 21.0; two-way use CRISPR/Cas9 system to produce genetic mutations in the Tas2rs gene cluster, including Tas2r106, Tas2r104, Tas2r105, and Tas2r114.
One sgRNA was designed for each Tas2rs (Figure 1A).A mixture consisting of Cas9 mRNA and four sgRNA mRNAs was microinjected into the cytoplasm of the zygotes.Sixty-eight embryos were injected, and nine mice were born from the 60 embryos transferred (i.e., 15% livebirth rate).PCR and T7EN1 assays were used to screen genetic mutation at the target sites.The PCR results indicated that six of the nine founders (66.7%) harbored genetic mutation in at least one targeted gene (Figure 1B).Two of them (6 and 7) were males, and the other four (1, 3, 5, and 8) were females.Amplification products were cloned and sequenced for further confirmation (Figure 1C).To study whether the mutation was heritable, four founders (3, 6, 7, and 8) were crossed with C57BL/6 mice.Genotyping results of F2 animals showed that all founders (3, 6, 7, and 8) passed the mutation to their offspring and produced four lines of Tas2rs-modified mice (Figure S1).Of these, one line was triple edited (8), and two lines were double edited (6 and 7).Our results also revealed that founder 8 was genetically mosaic (Figure 1B; Figure S1J), which was consistent with previous reports that most of the mutants that develop from the injected or electroporated zygotes were genetically mosaic. 28,38quencing results revealed that the triple-edited line (8) had  remarkable and selective loss of responses to 0.9 μM CYX.We also investigated the responses of mutant mice (Gnat3 −/− and Gnat3 −/− -Tas 2r104 −/− /Tas2r105 −/− ) to taste compounds.Gnat3 −/− mutant mice lost perception not only to low (30 mM) and high (100 mM) concentration sucrose but also to 30 mM glutamine and 0.9 μM CYX (Figure 4B).
Previous studies have revealed that ethanol may be involved in the perception of oral adaptation to bitter-tasting stimuli. 41,42To further study the taste perception of the Tas2rs mutant mice, different concentrations of ethanol were tested using two-bottle preference tests.WT mice showed the expected preference for all concentrations of ethanol.Like these controls, the double-(Tas2r104 −/− /Tas2r105 −/ ) and triple-(Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −− ) mutant mice also preferred any ethanol concentration (Figure 4D).
To further investigate the response of other bitter tastants, we tested these mice with different concentrations of quinine (QHCI).
The mutant and the WT mice strongly avoided QHCI, but the preference score in the mutant mice was higher than that in the WT mice for 10 μM QHCI (Figure 5D,E), indicating that their sensitivity was greatly decreased compared to WT mice in this concentration.
Subsequently, the mutant and the WT mice strongly avoided CYX, but the preference score in the mutant mice was higher than that in the WT mice for 1.2 μM CYX (Figure 5D,E).This indicated that QHCI pre-intake may increase the threshold sensitivity of bitter tastants, but perception for CYX still decreased in these mutant mice.These mutant mice of Tas2rs gene can still detect and avoid high concentrations of CYX.

| Two-bottle preference tests: QHCI, DB, and CuB
As the Tas2rs mutant mice unexpectedly showed preferences for 10 mM quinine, we then considered if they altered response to general bitter sensory experience.Consequently, Tas2rs mutant mice, double mutant mice, and WT mice were tested with QHCI and DB.
DB and QHCI have structurally dissimilar stimuli, and they stimulate different subsets of rat taste receptor cells (TRCs). 43,44The preference ratios for 10 μM QHCI were significantly higher for the mutant mice than for the WT mice (Figure 5F).DB solutions of varying concentrations were presented to mutant and WT mice to test for genotype differences in preference ratios (Figure 6A).The analyses of these data showed that Tas2r105 −/− /Tas2r114 −/− and Gnat3 −/− -Tas 2r104 −/− /Tas2r105 −/− mice had significantly higher preference scores for 0.5 and 1 mM DB solution than Tas2r104 −/− /Tas2r105 −/− /Tas2r11 4 −/− and WT mice (Figure 6B).In a heterologous cell expression system, CuB had been identified as cognate agonists for mTAS2R105 and mTAS2R114. 25 hypothesized that if two taste receptors contribute to CuB preference, the mutant mice (Tas2r104 −/− /Tas2r105 −/− and Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− ) would not detect CuB and would show preference ratios similar to that of water.These mutant mice were given various concentrations of CuB, and their preference ratios were analyzed.The analysis of the CuB intake data indicated that the preference ratios for the mutant mice were significantly larger at 0.9 and 1.8 μM CuB relative to those of the WT mice (Figure 6C,D).

| DISCUSS ION
A previous study has shown that muroid cluster I contains Tas2r107, Tas2r106, Tas2r105, Tas2r114, and Tas2r104, which have a common ancestor with human TAS2R10. 18Genetic deletion of Tas2r105 eliminates more neural responses and behavioral aversion to CYX.
But taste perception for denatonium, 6-n-propyl thiouracil, and quinine was not changed in Tas2r105 null mice. 40Recently, bitter taste signaling pathway is found in a large number of tissues and organs, and is intensively involved in biological functions in extraoral tissues.the 27-kb genomic fragment between two sgRNAs of Tas2r135 and Tas2r126. 49Furthermore, it has been frequently reported that the CRISPR/Cas9 technique can produce large somatic and heritable deletions of genomic fragments. 50Therefore, the current success in developing multiple Tas2rs KO mice suggests that it is feasible for developing multiple Tas2r KO mice using the CRISPR/Cas9 technique.
Most of the murine Tas2r genes were distributed in eight loci. 17,18us far, the regulation mechanism of Tas2rs gene expression remains unclear.Due to the altered expression of Tas2r136 and Tas2r137 in large airways in triple Tas2r KO mice (Tas2r143/Tas2r135/Tas2r126), the expression of Tas2rs may be regulated in a receptor type-dependent manner in the airway. 49The Tas2r104/Tas2r105/Tas2r114 genes are organized as a head-to-tail array within a single 6-kb DNA fragment. 20A previous study has suggested that shared regulatory sequences may coordinately regulate the expression of Tas2rs gene cluster. 20In this study, a deletion of interval sequence (817 bp) between Tas2r104 and Tas2r105 genes indeed affected the expression of mTas2rs in taste buds in the triple mutant mice.Meanwhile, we also observed the changed expression of Tas2rs in taste buds from double mutant mice.This further contributed to a different change in bitter taste perception.It is worth noting that the Tas2r104/Tas2r105/ Tas2r114 genes are often coexpressed in many extraoral tissues with the characteristics of tissue-specific expression. 7,9,24,51Thus, the expression regulation of this gene cluster in extraoral tissues still needs to be further studied.We speculate that these mutant mice, espe- for instance, is low in lingual epithelia but much higher in testis, 24 gut, and kidney. 7,21It has shown that progesterone can stimulate Tas2r114 and Tas2r110 in HEK293T cells stably expressing Ga16gust44 system, 25 indicating that hormones could function as Tas2r activators.
Thus, our Tas2r KO mice should be a valuable means to evaluate the role of Tas2r106/Tas2r104/Tas2r105/Tas2r114 gene cluster and allow us to check whether these TAS2Rs are really involved in the physiological functions in extraoral tissues and organs, such as lung, testis, gut, and kidney.studies may be critical for comprehending the role of these TAS2Rs in health and diseases.
We further measured the taste choices for these genetic mutants using two-bottle preference assays.As expected, the genetic mutation/deletion of Tas2r105 indeed eliminated the taste response to CYX but not to alcohol, sweet, umami, salty, and sour tastants.A further study also found that the genetic mutation of Tas2r106 did not show a selective impairment in their ability to taste CYX.The heterologous expression analysis using the Ga16gust44 cell system also failed to identify any agnoists for Tas2r106. 25In this study, a 13-or 2-bp deletion in Tas2r105 and a 4-bp deletion in Gnat3 can cause a frameshift, adequately destroying its function.Thus, a 2-bp insertion in Tas2r106 was more likely to disrupt its function, indicating that Tas2r106 may not be involved in the taste perception for CYX.
Another finding is that these Tas2rs mutant mice showed not only a changed perception ability for 10 μM QHCI and DB (0.5 and 1.0 mM) but also a reduced perception ability for CuB (0.9 and 1.8 μM).Tas2r105 −/− /Tas2r114 −/− double mutant mice can discriminate 0.5 mM DB, but Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− triple mutant mice cannot.This indicated that the genetic mutation of three bitter receptors can result in a loss of sensitivity for 0.5 mM DB. qRT-PCR results further showed that the deletion of interval sequence between mTas2r104 and mTas2r105 can alter the expression level of many mTas2rs genes in taste buds, for instance, downregulating the expression of mTas2r135 and mTas2r140, which may contribute to loss of sensitivity for DB. 25 In this study, two Tas2rs mutant mice showed an inconsistent taste perception to QHCI and DB, suggesting that two bitter substances can be sensed by a different set of receptors and/or a different subset of TRCs, which is consistent with previous reports. 43,44B is one of the derivatives of cucurbitacins, including the triterpenoid structure, which serve significant bioactivities such as anti-inflammatory effects, anticancer effects, antioxidative stress, and preventive effects against hepatotoxicity. 52,53CuB can induce plasma GLP-1 and insulin release in diabetic mice, which was involved in the activation of adenosine 5'-monophosphate (AMP)activated protein kinase through α-gustducin and Gβγ-signaling of taste receptors. 54Gustatory receptor is required for the taste sensation of CuB, which can activate Gr33a bitter-sensing gustatory receptor neurons in Drosophila melanogaster. 55Using a heterologous HEK293T-Gα16gust44 cell system, Tas2r105 and Tas2r114 were specifically activated by CuB. 25 In this study, these mice, including the mutation of mTas2r105 and mTas2r114, were not able to sense 0.9 and 1.8 μM CuB.Furthermore, the Gnat3 −/− -Tas2r10 Abbreviation: qRT-PCR, quantitative real-time polymerase chain reaction.

3 . 1 |
analysis of variance was used to compare multigroup variables, and Student's t-test was used to compare two independent groups.Statistical results are provided in the figure legends for each data set.Statistical significance was set at p < 0.05. 3 | RE SULTS Precise deletion of bitter taste receptors using multiple sgRNAs and heritability of dual bitter taste receptor mutation Most of the Tas2rs genes are located on the same chromosome in separate clusters defined by their physical locations.It is difficult to produce multiple Tas2rs gene KO using conventional gene targeting technology.Recent studies have shown that CRISPR/Cas9 technology is an efficient genome editing system.Therefore, we tried to F I G U R E 1 Genetic mutations of Tas2rs and Gnat3 mediated by sgRNA/Cas9 mRNA mixtures.(A) Target sites of the four sgRNA at the Tas2r106/Tas2r104/Tas2r105/Tas2r114 gene cluster and one sgRNA at the Gnat3 gene locus.Black rectangles represent exons of Tas2rs and Gnat3 genes.Target sites of SgRNA are shown by sequences.Protospacer adjacent motif (PAM) sites are underlined.(B) Representative electrophoretogram of F0 mice genotyping.Genome modifications were screened using PCR (polymerase chain reaction) and T7EN1 assays at the target sites in these founders.The number in the center indicates the size of PCR and digestion products; the red triangle indicates the occurrence of nucleotide deletion.(C) Sequencing of mutant alleles present in the F0 founders.Red color indicates the PAM sites; =, deletion; ^, insertion; WT, wild-type control.

a 9 - 3 . 2 |
bp insertion in Tas2r114 locus and a large deletion(2027 bp)   between the target sites of Tas2r104 sgRNA and Tas2r105 sgRNA (Figure1C).Thus, both TAS2R104 and TAS2R105 protein sequences were disrupted in this triple-edited mice.But a large deletion may generate a fusion gene, which contained 447 bp of Tas2r105 and 156 bp of Tas2r104, and intact 5′UTR of Tas2r105 gene and 3′UTR of Tas2r104 gene.The 9-bp insertion in Tas2r114 leads to a short inframe shift, so TAS2R114 may still be functional in this line.The double-edited line(6) showed a large fragment deletion of 478 bp in Tas2r104 around its sgRNA location and a 13-bp deletion in Tas2r105 around the sgRNA location.The 13-bp deletion in Tas2r105 creates a frameshift, consequently disrupting its function.A 478bp deletion causes a 157-amino acid deletion in TAS2R104, which might result in a frameshift in the protein sequence.In the second double-edited line(7), both the 10-bp deletion in Tas2r114 and the 3-bp deletion, 1-bp insertion in Tas2r105 causes a frameshift, consequently disrupting their function.In the third line, a 2-bp insertion may cause a frameshift in Tas2r106 (Figure1C).To block bitter taste signal pathway, we also developed Gnat3 mutant mice using CRISPR/Cas9 technology.One specific sgRNA for Gnat3 gene was designed.Gnat3 founder mice were obtained using the aforementioned procedure.A 4-bp deletion was confirmed by T7EN1 assays and sequencing (Figure1C).Then, founder mice were crossed with C57BL/6 mice, passed the mutation to their offspring, and gave rise to Gnat3 mutant mice.After at least six rounds of upgrading with C57BL/6 mice, Gnat3 +/− heterozygote mice were intercrossed to obtain homozygote mice.Gnat3 −/− mice were crossed with Tas2r104 −/− /Tas2r105 −/− mice.After several rounds of crossbreeding, we finally obtained double-KOGnat3 −/− -Tas2r104 −/− /Tas2r105 −/− mice.Mutation of bitter taste receptors did not affect the expression of Plcβ2 and Gnat3 in taste budsAfter successfully developing several Tas2rs KO and double-KO mouse models, including Tas2r104 −/− /Tas2r105 −/− , Tas2r105 −/− /T as2r114 −/− , Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− , Tas2r106 −/− , and Gnat3 −/− -Tas2r104 −/− /Tas2r105 −/− , we hoped to further investigate the effect of genetic mutation on taste-related gene expression.Taste GPCRs specific for sweet or bitter are expressed in any given type II cell separately.1 It is well known that representative molecular markers for type II taste cells are Gnat3 and Plcβ2.39First, we measured the expression of Gnat3 and Plcβ2 in taste buds from these mutant mice using indirect fluorescent antibody assay.Immunostaining with GNAT3 antibody showed that the expression of GNAT3 (Figure2A-D) in taste buds from mTas2rs mutant mice was similar to that of WT mice.But the expression of GNAT3 (Figure2E) was deleted in taste buds from Gnat3 −/− mutant mice, indicating that a 4-bp deletion can cause a frameshift in Gnat3 gene.We observed a similar expression pattern of PLCβ2 in taste buds from Tas2rs mutant and Gnat3 −/− -Tas2r104 −/− /Tas2r105 −/− mice with that of WT mice (Figure3A-D).PLCβ2-positive cells were also counted in taste buds in mutant and WT mice, but no significant difference was observed among them (Figure3E).F I G U R E 2 GNAT3 expression in theTas2rs and Gnat3 mutant mice.Microscopic images of frozen sections of taste bud-containing sections from circumvallate papillae from (A) WT (wild-type) and (B-E) mutant mice obtained using indirect immunofluorescence with specific antibodies directed against GNAT3.(A) WT, (B) Tas2r104 −/− /Tas2r105 −/− mice, (C) Tas2r105 −/− /Tas2r114 −/− mice, (D) Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− mice, and (E) Gnat3 −/− mice.Scale bar: 50 μm.Then, we employed qRT-PCR to study whether the genetic mutation of Tas2r105 cluster affects the expression level of other Tas2rs genes.The expression level of 19 mTas2rs in Tas2r105 −/− /Tas2r114 −/−mice was significantly different from that in WT mice (Figure3F).In Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− mice, the expression level of 18 mTas2rs was significantly changed (Figure3F); 17 mTas2rs in Gnat3 −/− -Tas2r104 −/− /Tas2r105 −/− mice were found to be significantly different compared to those in WT mice.In three mutant mice, several mTas2rs, including mTas106, mTas120, mTas121, mTas131, and mTas135, were significantly downregulated.mTas126 and mTas137 were significantly downregulated in Tas2r105 −/− /Tas2r114 −/− and Tas2r104 −/− /Tas2r105 −/− /Tas2r114 −/− mice.But the expression level of Tas2r114 and Tas2r110 was significantly upregulated in two mutant mice.In addition, we observed the expression of Tas2r104/105 fusion gene, produced by a 2027-bp deletion between the target sites of Tas2r104 sgRNA and Tas2r105 sgRNA.

E 3
Genetic mutation of mTas2rs has no effect on the PLCβ2-positive cell number in taste buds but can affect the expression profile of mTas2rs family.(A-D) Representative image of immunofluorescence staining with anti-PLCβ2 in taste buds.Scale bar: 100 μm.(E) PLCβ2-positive cell count in taste buds from these mutant mice.(F) The changed expression profile of mTas2r family revealed using qRT-PCR (quantitative real-time polymerase chain reaction) analysis.Statistical significance of differences between WT (wild-type) and Tas2rs mutant mice was assessed using Student's t-test, *p < 0.05.
The double mutant mice (Gnat3 −/− -Tas2r104 −/− /Tas2r105 −/− ) still failed to discriminate higher concentrations of CuB (2.4 and 3.0 μM) from water (Figure6C,D).The current results suggest that the mutation of Tas2r105 and Tas2r114 indeed attenuated the taste perception to CuB and bitter taste signal pathway contributes to CuB perception.

4
−/− /Tas2r105 −/− mice, due to the lack of GNAT3 and bitter taste receptors, did not respond to higher concentrations of CuB (2.4 and 3.0 μM).These results collectively demonstrate two points.First, Tas2r105 and Tas2r114 are indeed involved in the perception to CuB, but there may still be any unidentified chemoreceptor category to respond to CuB, because Tas2r105 −/− /Tas2r114 −/− mutant mice can still respond to higher concentrations of CuB.Second, taste perception to CuB needs the Gnat3-mediated receptor and its signaling pathway in mice.Briefly, the CRISPR/Cas9 technique can effectively knock out multiple Tas2rs genes at once.Although genetic mutation of the these Tas2rs genes did not affect the expression of GNAT3 and PLCβ2, the expression profile of Tas2rs genes was changed in taste buds.Two-bottle preference tests revealed that these mutant mice exhibited a decrease response to bitter tastants, including CYX, QHCI, DB, and CuB.These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many other extraoral tissues.E TH I C S S TATEM ENTThe study was approved by the Institute of Animal Use and Care Committee of Shanghai Public Health Clinical Center (GW2018-A055-01, GW2023-A030-01).O RCI D Feng Li https://orcid.org/0000-0003-1291-5315R E FE R E N C E S