Price, E, Gianfrancesco, O, Harrison, PT, Frank, B, Bubb, VJ and Quinn, JP (2021) CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region. International Journal of Molecular Sciences, 22 (4). ISSN 1422-0067

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Abstract

SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

Item Type:	Article
Uncontrolled Keywords:	Science & Technology; Life Sciences & Biomedicine; Physical Sciences; Biochemistry & Molecular Biology; Chemistry, Multidisciplinary; Chemistry; retrotransposon; SVA; cis-regulatory element; TRPV1; TRPV3; CRISPR; gene expression; Animals; Primates; Humans; Mice; DNA, Intergenic; Gene Expression; Gene Expression Regulation; Short Interspersed Nucleotide Elements; Alu Elements; Minisatellite Repeats; Genes, Reporter; TRPV Cation Channels; Promoter Regions, Genetic; HEK293 Cells; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; SVA; TRPV1; TRPV3; cis-regulatory element; gene expression; retrotransposon; Alu Elements; Animals; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; DNA, Intergenic; Gene Expression; Gene Expression Regulation; Genes, Reporter; HEK293 Cells; Humans; Mice; Minisatellite Repeats; Primates; Promoter Regions, Genetic; Short Interspersed Nucleotide Elements; TRPV Cation Channels; 0399 Other Chemical Sciences; 0604 Genetics; 0699 Other Biological Sciences; Chemical Physics
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QP Physiology R Medicine > RM Therapeutics. Pharmacology
Divisions:	Psychology (from Sep 2019)
Publisher:	MDPI AG
SWORD Depositor:	A Symplectic
Date Deposited:	11 May 2022 11:01
Last Modified:	11 May 2022 11:01
DOI or ID number:	10.3390/ijms22041911
URI:	https://researchonline.ljmu.ac.uk/id/eprint/16800

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