Locus-associated R-loop repression in cis

Abstract

R-loops exert varied beneficial or detrimental effects. To assess the function of an R-loop at a specific genetic locus, we had developed an inducible RNaseH1-EGFP-dCas9 (RED) protein chimaera as part of a locus-associated R-loop-modulating system (LasR). LasR is compatible with R-loop modulation in trans, which targets RED to one locus to repress R-loops at another spatially proximal site. Here we use the LasR system for R-loop modulation in cis, which consists of targeting RED directly to an R-loop. The combination of LasR in cis and trans will be essential to ascribe functions to specific R-loops within varied molecular contexts and study designs.

Protocol to use RNaseH1-based CRISPR to modulate locus-associated R-loops

“…If assessing R-loop modulation in trans , as in the case of the rDNA shown in this protocol, include qPCR primers for both the targeted locus (i.e., IGS28) and the R-loop-containing locus modulated in trans (i.e., IGS18) ( Abraham et al., 2020 ). If assessing R-loop modulation in cis , as in the case of the β-ACTIN locus shown elsewhere ( Khosraviani et al., 2021 ), include the qPCR primers that generate PCR amplicons that are within the vicinity of the sgRNA sites as summarized further below. Cell culture and RED/dRED induction conditions can be performed as presented herein previously.…”

“… (D and E) DRIP analysis shows that using RED (D) or dRED (E) together with sgIGS28 does not alter the levels of R-loops at unrelated genetic loci. (F) Schematic summarizing the pools of sgRNAs tested so far with this LasR system protocol when applied in trans at rDNA as shown previously ( Abraham et al., 2020 ) and herein or used in cis at the 5′Pause site of the single-copy β-ACTIN locus as shown elsewhere ( Khosraviani et al., 2021 ). Testing different pools of sgRNAs targeting both DNA strands allowed for the combination of three sgRNAs that specifically target to the locus of interest and enriched the RED and dRED chimaeras in ChIP at the targeted loci.…”

“…Such loops have been extensively studied using chromosome conformation capture, chromatin immunoprecipitation, and genome-wide sequencing ( Kempfer and Pombo, 2020 ). Second, the RED or dRED chimaera can be targeted directly to an R-loop-harboring locus for R-loop modulation in cis ( Figure 1 ) ( Khosraviani et al., 2021 ). The protocol described here is used for LasR targeting to highly repetitive ribosomal DNA repeats (rDNA) ( Abraham et al., 2020 ; Khosraviani et al., 2021 ).…”

“…Second, the RED or dRED chimaera can be targeted directly to an R-loop-harboring locus for R-loop modulation in cis ( Figure 1 ) ( Khosraviani et al., 2021 ). The protocol described here is used for LasR targeting to highly repetitive ribosomal DNA repeats (rDNA) ( Abraham et al., 2020 ; Khosraviani et al., 2021 ). While we have applied this system to achieve human R-loop control, we expect that analogous systems would be equally helpful to the study of R-loops in other species amenable to RNase H1-dependent RNA-DNA control and CRISPR-based genome editing.…”

“…To apply the LasR system for R-loop modulation in cis , first test single or pools of sgRNAs targeting the R-loop and its flanking regions. The LasR system can also be applied in cis as previously used to modulate R-loops at the single-copy human β-actin locus ( Khosraviani et al., 2021 ).…”