ACTIVATION OF TASTE RECEPTOR GENES IN MAMMALIAN CELLS USING CRISPR-CAS-9

Abstract

Suggested is a method for enhancing the expression of taste related receptor genes encompassing the following steps: (i) providing a culture of mammalian cells, the genome of said cells comprising at least one sweet receptor domain; (ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 30 nt guide sequence of said sgRNA being complementary to stretches within the non-coding and/or putative regulatory region upstream of the translation start codon of at least one sweet receptor gene; (iii) preparing a vector comprising an expression cassette encompassing at least one optionally modified CRISPR-Cas9, preferably CRISPR-dCas9VP64, and at least one optionally modified sg-RNA optionally containing aptamer structures for binding activator proteins; (iv) transfecting said culture of mammalian cells with said vector to target the genome for the presence of a DNA sequence that is complementary to the 10 to 30 nt guide sequence of said sgRNA; and (v) measuring the transcriptional enhancement of the sweet receptor mRNA by quantitative RT-PCR.

Claims

1. A method for enhancing the expression of taste related receptor genes encompassing the following steps: (i) providing a culture of mammalian cells, the genome of said cells comprising at least one sweet receptor domain selected from T1R1, T1R2, T1R3 or at least one domain of G-protein -gustducin (GNAT3); (ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 30 nt guide sequence of said sgRNA being complementary to stretches within the non-coding and/or putative regulatory region upstream of the translation start codon of at least one sweet receptor gene; (iii) preparing a vector comprising an expression cassette encompassing at least one CRISPR-dCas9VP64, and at least one optionally modified sg-RNA optionally containing aptamer structures for binding activator proteins; (iv) transfecting said culture of mammalian cells with said vector to target the genome for the presence of a DNA sequence that is complementary to the 10 to 30 nt guide sequence of said sgRNA; and (v) measuring the transcriptional enhancement of the sweet receptor mRNA by quantitative RT-PCR, wherein the sgRNA of the step (ii) is selected from (a) sgRNA2.0-t1r1_1 to sgRNA2.0-t1r1_11, when the sweet receptor domain is T1R1, (b) sgRNA2.0-t1r2_3 to sgRNA2.0-t1r2_6, when the sweet receptor domain is T1R2, (c) Cluster 6: sgRNA2.0-T1R3_1, _5, _7, _8, Cluster 1: sgRNA2.0-T1R3_1, _3, _4, _5, Cluster 5: sgRNA2.0-T1R3_3, _4, _6, _7 or Cluster 2: sgRNA2.0-T1R3_4, _5, _6, _7, when the sweet receptor domain is T1R3, and (d) sgRNA2.0-gnat3_1 to sgRNA2.0-gnat3_7, when the sweet receptor domain is GNAT3.

2. A sgRNA2.0 according to any of the sequences sgRNA2.0-t1r1_1 to sgRNA2.0-t1r1_11, sgRNA2.0-t1r2_1 to sgRNA2.0-t1r2_6 and sgRNA2.0-gnat3_1 to sgRNA2.0-gnat3_7.

3. The sgRNA2.0 of claim 2 recruited at least with one transcription factor activator.

4. The sgRNA2.0 of claim 2, wherein said at least one transcription factor activator is selected from the group consisting of p65 and HSF1.

5. A cluster of sgRNA2.0-t1r3 selected from the group consist of a first cluster (Cluster 6) of sgRNA2.0-t1r3 consisting of sgRNA2.0-T1R3_1, _5, _7, _8, a second cluster (Cluster 1) of sgRNA2.0-t1r3 consisting of sgRNA2.0-T1R3_1, _3, _4, _5, a third cluster (Cluster 5) of sgRNA2.0-t1r3 consisting of sgRNA2.0-T1R3_3, _4, _6, _7 and a fourth cluster (Cluster 2) of sgRNA2.0-t1r3 consisting of sgRNA2.0-T1R3_4, _6, _7.

6-8. (canceled)

9. A Cas9 or dCas9 protein loaded with (a) at least one of the sgRNA2.0 sequences according to claim 2.

10. A fusion protein of Cas9 or dCas9 protein and VP64 loaded with (a) at least one of the sgRNA2.0 sequences according to claim 2.

11. An expression cassette comprising (a) at least one Cas9, modified Cas9 or dCas9 and (b1) at least one sgRNA2.0 according to claim 2.

12. The expression cassette of claim 11, additionally comprising VP64, MS2, p65 and/or HSF-1.

13. The expression cassette of claim 11, additionally comprising T1R2, T1R3 and/or GNAT3.

14. A vector comprising the expression cassette of claim 11.

15. A screening method for identifying novel taste modulators comprising using the method of claim 1.

16. The method of claim 1, wherein the mammalian cells are primary human taste cells.

17-22. (canceled)

23. The method of claim 1, wherein the sgRNA is a sgRNA2.0 with a targeting sequence on the 5-end and one or several aptamers for recruiting transcription activator factors.

24. The method of claim 28, wherein said sgRNA2.0 recruits at least one transcription activator factor.

25. The method of claim 24, wherein said transcription activator factors encompass p65 and/or HSF1.

26. (canceled)

27. The method of claim 1, wherein said vector comprises an expression cassette additionally comprising VP64, MS, p65 and/or HSF-1.

28. The method of claim 23, wherein said aptamers are MS2 aptamers.

29. A Cas9 or dCas9 protein loaded with at least one cluster of sgRNA2.0 according to claim 5.

30. A fusion protein of Cas9 or dCas9 protein and VP64 loaded with at least one cluster of sgRNA2.0 according to claim 5.

31. An expression cassette comprising (a) at least one Cas9, modified Cas9 or dCas9 and (b2) at least one sgRNA2.0 cluster according to claim 5.

Description

SHORT DESCRIPTION OF THE FIGURES

[0054] FIG. 1A: Target location of the sgRNA-sequences in the non-coding upstream region of the indicated genes. 1B: Vector map comprising cas9 cassette; 1C: structure of sgRNA2.0 sequence (U6: human RNA polymerase III promoter sequence; S: spacer sequence from non-coding upstream region of the respective human T1R gene: MS2: RNA aptamer sequence)

[0055] FIG. 2A: Fold change of mRNA expression of the indicated genes compared to wild type/with vectors comprising indicated sgRNAs. 2B: Fold change of mRNA expression of the T1R3 gene compared to wild type/with vectors comprising different sgRNAs. The clusters have the following meaning (numbers refer to FIG. 1A): Cluster 6: sgRNA2.0t1r3_1, _5, _7, _8; Cluster 1: sgRNA2.0t1r3_1, _3, _4, _5; Cluster 5: sgRNA2.0t1r3_3, _4, _6, _7; Cluster 2: sgRNA2.0t1r3_4, _5, _6, _7

[0056] FIG. 3: Stimulation with saccharin and receptor-specific response in cells transfected with different sgRNA clusters (as explained in example 3). In wild type cells the T1R2/T1R3 genes are hardly active and therefore the heterodimeric sweet receptor is expressed on a very low level.

[0057] FIG. 4A: Fold change of mRNA expression of the Tas1R2 and Tas1R3 genes in HTC cultivated in different media (as indicated) after transduction with adenoviral vectors expressing dCas9VP64, MS2-p65-HSF1 and four different sgRNA2.0 with specific combination of guide sequences in tandem orientation: gnat3_1, t1r3_4, t1r3_6, t1r2_3 (tandem_sweet). The fold change was normalized against the expression level of the target genes in HTC transduced with dCas9VP64, MS2-p65-HSF1 and empty sgRNA2.0 control adenoviral vectors. 4B: 10 l aliquots of qRT-PCR reactions performed with total RNA prepared from HTC transduced with adenoviral vectors expressing CRISPR-dCas9, VP64, sgRNA2.0, MS2, p65 and HSF1 and either tandem_sweet (expressing the four sgRNA2.0 as in 4A) or empty sgRNA2.0 control (without specific guide sequences), respectively, were separated on 1% agarose gel. The T1R3 and TOP1 amplicons are indicated.

EXAMPLES

Example 1

[0058] Transcriptional activation of T1R1-, T1R2-, T1R3- and -gustducin (GNAT3)-mRNA by transfection of HEK293 cells with a vector comprising an expression cassette consisting of CRISPR-dCas9, VP64, sgRNA2.0, MS2, p65 and HSF1. 7 to 12 different species of sgRNAs were designed to target specific stretches of the non-coding region of the T1R1, T1R2, T1R3 or -gustducin (GNAT3) gene, respectively. FIG. 1A shows the target location of the sgRNA-sequences in the non-coding upstream region of the indicated genes.

[0059] Transcriptional enhancement was quantified by quantitative RT-PCR as shown in FIG. 2A. Provided is the fold change of mRNA expression of indicated genes compared with vectors comprising different sgRNAs relative to wild type cells transfected with vectors without specific guide sequences. Depending on the binding site of the activators up to 250, 300 or 50-fold increase of T1R1, T1R2 or G-protein gustducin mRNA, respectively, could be achieved. In contrast, no significant transcriptional enhancement of the T1R3-mRNA was observed with any of the 12 T1R3-constructs. However, transcript level enhancement of the T1R3-mRNA, ranging from 200 to 1200-fold, was obtained by co-transfection of the cells with 4 different T1R3-specific vectors (FIG. 2B). The clusters have the following meaning (numbers refer to FIG. 1A):

Cluster 6: sgRNA2.0-T1R3_1, _5, _7, _8
Cluster 1: sgRNA2.0-T1R3_1, _3, _4, _5
Cluster 5: sgRNA2.0-T1R3_3, _4, _6, _7
Cluster 2: sgRNA2.0-T1R3_4, _5, _6, _7

Example 2

[0060] Functional assay for the heterodimeric sweet receptor T1R2/T1R3 in HEK293 cells transfected with different vectors comprising dCas9-VP64, MS2-p65-HSF1, sgRNA2.0 specific for T1R2, T1R3, and gustducin alpha (GNAT3). lonomycin was used as -unspecific-positive control for receptor activity in the Fluo-4 assay monitoring intracellular calcium concentration. Response to ionomycin was set as 100%. Stimulation with saccharin shows a clear receptor-specific response in cells transfected with sgRNA clusters, whereas in the wild type cells the T1R2/TR3 genes are hardly active and therefore the heterodimeric sweet receptor is expressed on a very low level (FIG. 3).

Vector combination 1: 4cas9, 4sgRNA2.0 (GNAT3_1, T1R2_3, T1R3_4, T1R3_6)
Vector combination 2: 4cas9, 4sgRNA2.0 (GNAT3_2, T1R2_4, T1R3_5, T1R3_6)

Example 3

[0061] Quantitative RT-PCR with human primary taste cells BR-HTC (WO2013/160415). Transcriptional enhancement was quantified by quantitative RT-PCR as shown in FIG. 4A. Provided is the fold change of mRNA expression of T1TR2 and T1R3 genes in HTC by transduction with adenoviral vectors expressing dCas9-VP64, MS2-p65-HSF1 and sgRNA2.0 with four different guide sequences targeting upstream regions of gnat-3, T1R2 and T1R3 promoters, respectively (U6-sgRNA2.0-gnat3_1, U6-sgRNA2.0-t1r3_4, U6-sgRNA2.0-t1r3_6, U6-sgRNA2.041r2_3). The mRNA level of HTC transduced with adenoviral vectors expressing dCas9-VP64, MS2-p65-HSF1 and empty sgRNA2.0 was defined as 1. Depending on the cultivation media (indicated in the FIG. 4A) up to 40 or 100-fold increase of T1R2 or T1R3 mRNA, respectively, could be achieved. Aliquots of the four qRT-PCR reactions were analysed by agarose gel electrophoresis (FIG. 4B). Consistent with the qRT-PCR data, the transcriptional enhancement of T1R3 in HTC transduced with dCas9-VP64, MS2-p65-HSF1 and tandem adenoviral vector (lane 5) could be detected compared to empty sgRNA2.0 vector control (compare lanes 5 and 6). In contrast, the expression level of the internal control gene TOP1 was not affected by adenoviral transduction (lanes 2 and 3). Thus, the designed sgRNA2.0 constructs and the method provided here, allows the simultaneous modulation of taste related genes in human taste cells.