Abstract
Recent work suggests extensive adaptation of transposable elements (TEs) for host gene regulation. However, high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made systematic interrogation of the regulatory contributions of TEs challenging. Here, we employ CARGO, our recent method for CRISPR gRNA multiplexing, to facilitate targeting of LTR5HS, an ape-specific class of HERVK (HML-2) LTRs that is active during early development and present in ~700 copies throughout the human genome. We combine CARGO with CRISPR activation or interference to, respectively, induce or silence LTR5HS en masse, and demonstrate that this system robustly targets the vast majority of LTR5HS insertions. Remarkably, activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of hundreds of human genes. These effects require the presence of retroviral sequences, but occur over long genomic distances, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in apes.
Highlights
Half of the human genome is composed of transposable elements (TEs), which are increasingly being recognized not just as parasitic DNA, but as an important source of regulatory innovation for the host (Chuong et al, 2017; Feschotte, 2008; Rayan et al, 2016; Thompson et al, 2016)
To investigate the role of HERVK LTR5HS insertions in the regulation of embryonic gene expression and, more broadly, to establish a proof of principle for using CARGO to simultaneously target hundreds of repetitive elements interspersed across the genome, we designed a CARGO array with 12 distinct guide RNA (gRNA) transcriptional units, altogether predicted to recognize ~91% (635/697) of LTR5HS integrations in the human genome when allowing zero mismatches between gRNA sequences and LTR5HS sequences (Figure 1—figure supplement 1)
At zero mismatches, ~87% of LTR5HS insertions are targeted by at least two gRNAs, and ~57% by at least four gRNAs (Figure 1—figure supplement 1), an important consideration given that a single gRNA is often insufficient for robust gene activation/ silencing by CRISPRa/CRISPRi (Cheng et al, 2013; Perez-Pinera et al, 2013)
Summary
Half of the human genome is composed of transposable elements (TEs), which are increasingly being recognized not just as parasitic DNA, but as an important source of regulatory innovation for the host (Chuong et al, 2017; Feschotte, 2008; Rayan et al, 2016; Thompson et al, 2016). Despite evidence suggesting the importance of LTRs and other TEs in rewiring gene regulatory networks, most current studies are either correlative or focus on the analysis of individual insertions, rather than on systematically perturbing specific TE classes, with one notable exception of a report utilizing transcription activator-like effector (TALE) fused to effector domains for functional perturbations of mouse LINE1 elements (Jachowicz et al, 2017) This knowledge gap is associated with technical challenges, as LTR subfamilies are often present in hundreds or thousands of copies, which are highly repetitive, but, due to accumulated mutations, sufficiently sequence-divergent to prevent their recognition by a single short-sequence-dependent factor, such as a zinc finger protein or CRISPR guide RNA (gRNA). Our work provides a novel and broadly applicable strategy for functional manipulation of specific TE classes across the genome and supports a pervasive role of LTRs as embryonic gene enhancers
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