CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region

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 TRPV1 and 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.

A SINE-VNTR-Alu at the LRIG2 locus is associated with proximal and distal gene expression in CRISPR and population models

“…Additionally, these two variables appear to be correlated. This study supports a growing body of evidence that SVA RIPs can influence expression of nearby genes 28 , 29 , and should therefore be regarded as potentially important regulatory inputs for local gene expression.…”

TRPV3 Ion Channel: From Gene to Pharmacology

“…Price et al identified that the trpv1/trpv3 intergenic region is enriched with humanspecific SINE-VNTR-Alu (SVA) retrotransposon insertions [20]. The SVA, located approximately 400 bp downstream the trpv1 3 UTR and 5.7 kb upstream of the 5 trpv3 transcriptional start site, serves as a cis-regulatory element for the trpv3 gene.…”

“…TRPV3 is expressed in sensory neurons such as the dorsal root ganglia (DRG) and in the trigeminal ganglia (TG) in humans and nonhuman primates [8]. In many human tissues, TRPV1, functionally significant in neuropathic pain conditions, and TRPV3, are co-expressed and can form heteromeric channels, which was not observed in mice [20]. Perhaps this underlies the key regulatory differences between the two species.…”

Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement

“…For a long time, CRISPR-based large fragment deletions have been used to study CREs in various cells . Using two sgRNAs to target a sequence causes the sequence between the two sgRNAs to be deleted. − Currently, CRISPR/Cas-based large fragment deletion in animals is used to study lncRNAs and CREs. − The improvement and application of this tool improved molecular breeding methods and broadened the application range of CRISPR toolsets (Supplementary Table 1).…”