Interrogating lncRNA functions via CRISPR/Cas systems

ABSTRACT

Long noncoding RNAs (lncRNAs) are an increasing focus of investigation due to their implications in diverse biological processes and disease. Nevertheless, the majority of lncRNAs are low in abundance and poorly conserved, posing challenges to functional studies. The CRISPR/Cas system, an innovative technology that has emerged over the last decade, can be utilized to further understand lncRNA function. The system targets specific DNA and/or RNA sequences via a guide RNA (gRNA) and Cas nuclease complex. We and others have utilized this technology in various applications such as lncRNA knockout, knockdown, overexpression, and imaging. In this review, we summarize how the CRISPR/Cas technology provides new tools to investigate the roles and therapeutic implications of lncRNAs.

INTRODUCTION

As only approximately 2% of the human genome harbors protein-coding genes, there is increased interest in the roles of non-coding regions of the human genome [1]. Using the latest sequencing technologies, various regulatory non-coding RNAs have been identified. These non-coding RNAs (ncRNAs) include small ncRNAs, which are <200 nucleotides (nts) in length, such as miRNAs and piRNAs, and long non-coding RNAs (lncRNAs), which are >200 nts long [2]. Despite being expressed at low levels, being poorly conserved, and showing tissue-specific expression [2,3], lncRNAs have been shown to play important roles in diverse cellular processes such as controling transcription, acting as molecular decoys, guiding localization, and providing protein scaffolding [3]. lncRNAs can be localized to the nucleus and/or the cytoplasm, exerting effects on DNA, RNA, and/or proteins. Due to low abundance, tissue-specific expression, a wide range of cellular roles, and limited research techniques, studying lncRNAs may be complex and challenging.

Often, to interrogate the function of a novel lncRNA, changes are induced to their expression. However, widely used tools such as RNA Interference (RNAi) and antisense oligonucleotides (ASOs) are less effective in lncRNA studies [4–6]. RNAi uses a multi-protein RNA-induced silencing complex (RISC) to induce RNA degradation mediated by small interfering RNA (siRNA) binding to the target RNA [7]. Though useful in studies of cytoplasmic molecules, RNAi is usually less effective in suppressing nuclear lncRNA molecules due to the predominantly cytoplasmic localization of RISC in most cell types. ASOs function by binding to RNA then selectively degrading the ASO-RNA hybrids via the RNase H1 enzyme, which recognizes DNA-RNA hybrids. The use of ASOs relies on exogenous modified oligonucleotides to produce transient effects, making this technique complex. The most significant limitation to these techniques is the resulting degradation of the lncRNAs , limiting their use in mechanism of action studies while not yielding complete loss-of-function.

Beginning in the early 2000s the widespread use of genomic manipulation techniques, such as Zinc Finger Nucleases (ZFNs), changed the scope of molecular biology. Soon after, the use of Transcription Activator-Like Effector Nucleases (TALENs) followed. Almost 20 years later, these techniques remain popular, and now have been utilized in lncRNA studies. For example, Lee et al. (2016) were able to use the TALENs technology, utilizing homology-directed insertion, to insert a Lox-Stop-Lox cassette containing tandem polyadenylation signals into the NORAD locus, silencing expression of the lncRNA [8]. While these systems have accomplished advanced genetic manipulations of lncRNAs, they continue to present issues regarding time, cost, and flexibility, as well as challenges regarding protein engineering, synthesis, and validation [9].

A more powerful method for genetic manipulation of lncRNAs is the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system. The CRISPR system is a genomic editing technique that has emerged as a revolutionary tool in molecular biology due to its ease and flexibility. Because it relies primarily on the use of gRNAs (gRNAs) to direct the catalytic activity of the Cas enzymes [10,11], it is preferable to ZFNs or TALENs that are more laborious. The Cas enzyme creates a double-strand break (DSB) that is often repaired by non-homologous end joining (NHEJ). While the CRISPR system traditionally uses Cas9, it can also be utilized with other enzymes such as dCas9, Cas12, and Cas13, offering great flexibility. Catalytically dead Cas9 (dCas9) disrupts gene function by blocking transcription. Cas12 has two orthologs with known functions; Cas12a targets single-stranded DNA (ssDNA) [12] while Cas12b targets double-stranded DNA [13]. Cas13 selectively targets single-stranded RNA (ssRNA) and has been utilized to edit RNA, image RNA, and knockdown transcripts via RNA degradation [14,15].

Due to its versatility and flexibility, the CRISPR system is especially useful in studies of lncRNAs. The technique presents the ability to target nuclear or cytoplasmic lncRNAs, disrupt the genome to selectively interact with (or avoid) adjacent genetic material, and upregulate or downregulate transcription, all without degrading the lncRNA which allows for advanced mechanism of action studies. The adaptability of the various Cas enzymes presents opportunities to target the gene locus as well as the RNA transcript. By having this flexibility, studies can better address the highly specific and versatile roles of lncRNAs. Although many of these techniques are recent developments, if selected properly, the CRISPR/Cas system can greatly advance our understanding of lncRNA function. In this review, we highlight the various uses of the CRISPR/Cas system in studying lncRNA functions.

1. Targeting lncRNA genes with Cas9

The most basic use of CRISPR/Cas9 is utilizing a gRNA to induce a nonsense or frameshift mutation via a DSB and NHEJ. While an effective method for disrupting proper protein production in protein-coding genes, these mutations are rarely significant enough to inactivate a lncRNA gene. Due to poor evolutionary conservation, it is often unclear which region of a lncRNA is the active domain, preventing efficient selection of gRNA targets. If able to select a target site on the lncRNA gene, it is important to consider off-target effects as well as implications for adjacent genes. lncRNAs may be intergenic or intragenic. Intragenic lncRNAs may overlap with protein-coding genes or may be located within the introns of host genes (intronic lncRNA). lncRNA promoters may be found within adjacent genes or shared with nearby protein-coding genes [16]. The span of lncRNA promoters across the genome poses significant limitations on the use of CRISPR knockout systems because targeting a lncRNA may have significant impacts on nearby genes [16]. As a result of these limitations, new uses of the CRISPR system have emerged. A summary of some of these tools is shown in Table 1.

Table 1.

CRISPR/Cas systems used for lncRNA studies

Editing System	Advantages	Disadvantages	Cas Variant	Approach	Example lncRNAs	Reference
CRISPR/Cas9	Targeted deletion/mutation of gene to decrease lncRNA expression	Risk impacting the expression of adjacent or related genes	Cas9	Deletion of whole lncRNA loci	PINCR, Gm26878	(Chaudhary et al., 2017), (Szafranski et al., 2017)
			Cas9	Deletion of transcription factor binding sites	PURPL	(Li et al., 2017)
			Cas9	Deletion of large fragment of lncRNA locus	Rian	(Han et al., 2014)
			Cas9	Genome-wide screen via genomic deletion of lncRNA loci	-	(Zhu et al., 2016)
			Cas9	Genome-wide screen via deletion of lncRNA splice sites	-	(Liu et al., 2018)
CRISPRa	Overexpress lncRNA genes without permanent alteration of genome	Risk impacting the expression of adjacent genes	dCas9-VP64	Overexpression	MALAT1, NEAT1	(Xie et al., 2017), (Yamazaki et al., 2018), (Butler et al., 2019)
			dCas9-VP64	Genome-wide screen via CRISPR activation	-	(Bester et al., 2018)
CRISPRi	Repress lncRNA expression without permanent alteration of genome	Risk impacting the expression of adjacent genes	dCas9-KRAB	Knockdown	DIRC3, roX1, roX2	(Coe et al., 2019), (Ghosh et al., 2016)
			dCas9-KRAB, KRAB-dCas9	Genome-wide screen via knockdown of lncRNAs	-	(Liu et al., 2017), (Cai et al., 2020)
CRISPR/Cas13	Directly target lncRNA molecule	Transcription of lncRNA still occurs	Cas13d	Knockdown	HOTTIP, MALAT1	(Konnerman et al., 2018)
		Can only target ssRNA	dCas13b	Imaging	NEAT1, SatIII	(Yang et al., 2019)
			Cas13a	Genome-wide knockdown of lncRNAs	-	(Xu et al., 2020)

1.1. Gene editing with Cas9

One approach to produce an effective lncRNA knockout is to use two paired gRNAs, targeting the beginning and end of the lncRNA gene to excise the full lncRNA locus. It has been found that introducing multiple gRNAs increased the efficiency of this type of knockout [17,18]. Many studies attempt to excise a large fragment of a lncRNA loci rather than the full lncRNA [17,19,20]. For example, Han et al. (2014) deleted up to 23 kb of Rian, a 57.8 kb lncRNA gene, successfully decreasing Rian expression in the offspring of female mice using a Cas9-mediated deletion [17]. Zhu et al. (2016) used this technique for a high-throughput screen, screening over 700 lncRNAs that have oncogenic or tumour suppressive activity [19]. They also found that the paired gRNA system was more efficient when the two gRNAs are introduced in a single lentivirus vector under two separate U6 promoters [19]. Some studies have used this technique to delete full lncRNA loci successfully [5,21,22].

Alternately, Cas9 may be targeted to a transcription factor binding site or promoter of a lncRNA. It has been proposed that the recruitment of transcription factors during lncRNA transcription may impact the transcription of nearby genes [23]. By mutating the promoter of a lncRNA, one can elucidate the effects of lncRNA transcription on adjacent genes, rather than impacts of the lncRNA transcript itself on neighbouring genes [24]. Engreitz et al. (2016) performed a study in which they successfully knocked out promoters of 12 lncRNAs using 2–3 gRNAs to assay for effects on the expression of adjacent genes [25]. They found that in 5 of the 12 lncRNAs, knocking-out the lncRNA promoter impacted genes approximately 5–71 kb from the targeted region [25]. A similar approach has been used to determine the roles of lncRNAs, including UCA1, P14AS, and PURPL, by knocking out transcription factor binding sites in their promoters [26–28]. This type of knockout has been found to be very effective, as demonstrated by Zhen et al. (2017) when they found an 80% reduction in UCA1 expression with a gRNA targeting the UCA1 promoter [26]. When knocking out the promoter-like sequence upstream of P14AS, Ma et al. (2020) found reduced P14AS transcription as well as decreased downstream ANRIL expression [27].

Another location that can be targeted to study lncRNAs are splice sites within the lncRNA gene. Targeting the splice site with ASOs has previously been shown to be effective in knocking down lncRNA expression [29]. Meanwhile, the CRISPR/Cas9 system has been utilized to target pre-mRNA splice sites on the DNA sequence, leading to alternative splicing and gene knockdown through nonsense-mediated decay [30,31]. By combining these techniques, gRNAs can target Cas9 to lncRNA splice sites to produce effective intron retention or exon deletion [32]. Liu et al. (2018) used this technique on a genome-wide scale to screen for essential lncRNAs [32]. It is significant to note that later studies suggest that targeted deletions using this approach may not be as effective as previously suggested [33]. Targeting lncRNA splice sites with CRISPR/Cas9 can be used both in loss of function studies as well as lncRNA screens. Though not commonplace, this method offers a solution to issues associated with deleting a large portion of the DNA while producing an efficient knockout with minimal off-target effects.

Lastly, knock-in models can be utilized to study how lncRNAs function in cells. Due to the large size of lncRNAs, knocking-in a lncRNA is not practical. Studies have knocked-in tags to the lncRNA gene to visualize lncRNA localization, using a system coined CRISPR-mediated Endogenous lncRNA Tracking (CERTIS) [34]. Alternately, Lee et al. (2016), utilizing TALENS, knocked-in a transcriptional stop element to inactivate the NORAD locus [8]. Yin et al. (2015) knocked-in a polyA stop cassette, producing a 55% decrease in Haunt1 lncRNA transcripts using CRISPR/Cas9 [18].

1.2. Challenges of Gene editing with Cas9

The major limitation to excising large fragments of lncRNAs using two gRNAs is the significant risk of impacting adjacent genes, such as those that may overlap with the lncRNA gene, those that are regulated by transcription of the lncRNA, or those with DNA elements in the lncRNA locus that may regulate other genes [35]. While targeting Cas9 to transcription factor binding sites, promoters, or splice sites minimizes the risk of deleting otherwise important genetic material, these techniques present issues of their own. When manipulating a lncRNA promoter there is still a possibility of impacting other genes, such as those regulated by the same promoter as the lncRNA or genes that may overlap with the targeted promoter. Regarding knock-ins, these types of studies require extensive research to determine potential off-target effects. The selection of the location where the knock-in will occur is crucial. Specific to lncRNAs, though, the bidirectional relationship that a lncRNA may have with adjacent genes must be considered when determining where to perform a knock-in. Overall, manipulations to the DNA encoding a lncRNA present issues in regard to disrupting surrounding genetic material due to the large size of lncRNAs as well as their overlap and interactions with other genomic elements.

1.3. Silencing or overexpression

Rather than permanently altering the DNA in a knockout or knock-in model, as described in the previous section, the CRISPR interference (CRISPRi) tool offers a method to repress gene expression without permanently mutating the genome. CRISPRi utilizes dCas9 and a gRNA to block RNA Polymerase II transcription, reversibly repressing the functional activity of the gene of interest [36,37]. This technology has been used in lncRNA screens [38,39] as well as silencing studies [40–42]. To further enhance gene repression, transcription repressors such as KRAB may be fused to the dCas9-gRNA system [43]. Despite the widespread use of KRAB in CRISPRi studies, it has been found that alternate transcription repressors, such as the SIN3-interacting domains derived from the MXD1 protein, may be more effective than KRAB in CRISPRi studies of lncRNAs [44].

After the invention of CRISPRi, CRISPR activation (CRISPRa) was developed utilizing dCas9 fused to RNA-polymerase recruiting factors [45]. Transcription activators such as VP64, p65, and Rta are commonly used to induce gene expression [16]. CRISPRa can be used in screens as well as overexpression studies [46]. This technique has been used to elucidate the functions of lncRNAs, such as MALAT1 and NEAT1 [47–49].

1.4. Challenges of the CRISPRi/CRISPRa system

CRISPRi and CRISPRa have similar advantages and disadvantages. First, by not disrupting the DNA sequence or causing any permanent alterations, these methods are less likely to impact neighbouring genes permanently. Nonetheless, the recruitment of transcription activators and repressors may cause changes in expression levels of adjacent genes. For example, when overexpressing lncRNA RP11-326A19.4 in HEK293T cells, Soubeyrand et al. (2019) saw increased expression of cytokine IL6, suggesting that CRISPRa has unpredictable off-target effects [50]. By utilizing transcription repressors or activators, the effects of lncRNA expression on nearby genes can be observed. This is significant as transcription of a lncRNA itself may regulate some adjacent genes. The CRISPRa system induces overexpression of the lncRNA isoforms in the ratio that they are naturally spliced, rather than a knock-in model that may only induce expression of specific isoforms. CRISPRi and CRISPRa may interact with nearby promoters, causing confounding results, a limitation that should be considered if the promoter of the lncRNA of interest is within another gene.

2. Targeting lncRNA molecules with Cas13

Recently, there has been an increase in the use of CRISPR/Cas13 to target RNA. The CRISPR/Cas13 system employs Cas13, an RNase which assembles with CRISPR RNA (crRNA), to form a programmable RNA targeting system. The Cas13 proteins contain two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains with endogenous RNase activity [51]. Presently, there are four known orthologs of the Cas13 protein (Cas13a, Cas13b, Cas13c, and Cas13d), each exhibiting different levels of activity in human cell lines [52]. This system has been utilized mainly in lncRNA knockdown [15,53] and imaging systems [54], although it has the potential to be used in a range of studies focusing on lncRNA function.

2.1. lncRNA knockdown with Cas13

Prior methods of lncRNA knockdown, such as RNAi or ASOs, have the potential to induce significant off-target effects [55–58]. Even CRISPRi can produce non-negligible off-target effects, inducing unintentional transcriptional changes in single-cell clones [59]. The CRISPR/Cas13 approach has been shown to have similar knockdown efficiency, as compared to RNAi, with fewer non-specific effects [60]. Because Cas13 can localize throughout the cell, it has the potential to knockdown both nuclear and cytoplasmic lncRNAs. Using CasRx, a Cas13d ortholog, Konermann et al. (2018) showed that the system allowed for specific, efficient knockdown of lncRNAs in human cell lines [15]. Xu et al. (2020) have also utilized the CRISPR/Cas13 system to perform a high-throughput screen with successful knockdown of very long intergenic non-coding (vlinc) RNAs [53]. This finding suggests that this system can be utilized for phenotypic assays of lncRNAs throughout the cell.

2.2. Challenges of the CRISPR/Cas13 System

Although the CRISPR/Cas13 system provides a robust method to target specific RNAs, it has some limitations. The system utilizes various orthologs of Cas13, each with limitations in their knockdown efficiency. This places restrictions on target locations and sgRNA design for certain model organisms depending on the ortholog. In vitro and bacterial studies using Cas13a have found that the system preferentially targets regions with Protospacer Flanking Sequences (PFSs) [61]. The Cas13b system has also been shown to display a preference for PFSs in bacterial cells [62,63], although the findings regarding PFS requirements have been contradictory in mammalian cells [64]. The secondary structure of the target lncRNA also limits the ability of the CRISPR/Cas13 system. Because Cas13 can only target ssRNA, the system has the potential to function well for lncRNAs without high-order structures [61]. Nevertheless, many lncRNAs do contain regions of high-order or double-stranded structures, complicating sgRNA design [64]. The challenge can be overcome by studying the structure(s) of the lncRNA(s) of interest. Bandaru et al. (2020) developed a workflow that uses Structure-Seq data to strategically determine appropriate sgRNA targets within single-stranded region(s) of a lncRNA, and Wessels et al. (2020) developed a program that predicts gRNA targets for Cas13 [64,65]. Even though there are challenges, the CRISPR/Cas13 system is a powerful and specific tool for knocking down or visualizing lncRNAs.

3. Functional characterization of lncRNAs using CRISPR

By properly selecting and executing the various CRISPR techniques, one can perform highly nuanced manipulations of lncRNAs beyond traditional knockouts, knockdowns, and knock-ins.

3.1. lncRNA imaging

Previously, the MS2-Capsid Protein (MS2-MCP) system has been used to visualize RNA [66]. However, this system uses aptamers that have the potential to disrupt RNA structure, expression, and function [54]. Wang et al. (2019) developed a system, entitled CRISPR live-cell fluorescent in situ hybridization (CRISPR LiveFISH) in which oligonucleotides bound to a fluorophore were used in combination with both dCas9 and dCas13 to visualize DNA and RNA in live cells [67]. While the system has not been tested in studies of lncRNA, it offers great promise for future studies. Meanwhile, Yang et al. (2019) visualized lncRNAs using a live-cell imaging method that uses a fluorescently labelled catalytically inactive Cas13 (dCas13) protein to visualize both nuclear and cytoplasmic RNAs [54]. By assaying for functional dCas13 orthologs, they determined that dPspCas13b and dPguCas13b were efficient dCas13 proteins that can label lncRNAs in mammalian cells [54]. This system was used to effectively determine the localization of the nuclear lncRNA, NEAT1. Dual-colour imaging was also performed using two dCas13 proteins to visualize two nuclear lncRNAs, NEAT1 and SatIII, concurrently. This technique allows for dual lncRNA tracking, which has the potential to be expanded to visualize lncRNAs in any cell compartment. The system may also be paired with dCas9 to visualize DNA and RNA simultaneously using a dual-colour system.

3.2. Determining if a lncRNA is cis- or trans-acting

In investigating the function of a lncRNA, it is important to identify its location within the cell. To determine the cellular localization of a lncRNA, subcellular isolation or dCas13 imaging can be performed as previously described [54]. Once the localization of a lncRNA of interest has been identified, one can determine whether the lncRNA is cis-acting or trans-acting. A cis-acting lncRNA regulates the expression and/or chromatin states of surrounding genes, while a trans-acting lncRNA regulates gene expression at a distance [68,69]. To determine whether a lncRNA acts in trans, a rescue experiment can be performed in CRISPR/Cas9 knockout cells. If there is a reversal of the knockout phenotype following the rescue experiment, then the lncRNA likely acts in trans [68]. lncRNA localization in the cytoplasm and/or nucleoplasm further supports that the lncRNA is trans-acting [69]. Nevertheless, it is important to note that if a lncRNA predominantly localizes to the cytoplasm, its coding potential should be interrogated (reviewed in [70]).

Investigating cis-acting lncRNAs may pose challenges due to the ambiguity between the function of the lncRNA and the locus from which it is transcribed [68]. However, the CRISPR/Cas system can provide insight into cis-acting lncRNA function. If the transcription of a lncRNA occurs at a singular promoter, CRISPR/Cas9 knockout of a small region within the first exon of the lncRNA can be performed, and, sequentially, the expression of nearby genes can be determined [68]. A major confound of this type of study occurs due to the direct manipulation of genomic DNA by the CRISPR/Cas9 system. The resultant phenotype may be due to alterations to the mature RNA or the lack of lncRNA transcription completely. A great example of this phenomenon is shown by the investigation of lincRNA-p21. After studying the expression of p21 in lincRNA-p21^−/- mouse embryonic fibroblasts (MEFs), lincRNA-p21 was classified as cis-acting because it was found to activate the expression of p21, a neighbouring gene [71]. Alternatively, a study by Groff et al. (2016) suggests that it is not the expression of lincRNA-p21 that mediates its role in the p53 signally pathway, but the DNA enhancer elements that are located within the lincRNA-p21 locus [35]. To better elucidate the function of lincRNA-p21, CRISPRi or CRISPRa techniques could be utilized to further study the effects of cis activation or the enhancer regions of the locus [72–74]. However, these methods may introduce unintentional changes in the chromatin environment which may be falsely attributed to the alterations in lncRNA transcription. Additionally, the lncRNA can be knocked down using CRISPR/Cas13, although the consequences of lncRNA transcription may be indistinguishable from the phenotypes observed as a result of the knockdown.

An alternate method to study lncRNA cis-effects is through tethering the transcript to neighbouring genes. Luo et al. (2016) utilized dCas9 and a gRNA fused to the lncRNA of interest to guide and tether the complex to a nearby locus, determining if the lncRNA regulated the expression of the targeted gene [75]. Another method for investigating lncRNA function is CRISPR-Display (CRISP-Disp) [76,77]. Shechner et al. (2015) engineered this technique to target large lncRNA cargos to endogenous loci [76]. This system uses gRNAs with integrated functional lncRNA domains and a dCas9 molecule fused to VP64 [77]. Shechner et al. (2015) found that the complexes were able to induce significant activation of endogenous loci. This system has the potential to be used for additional applications such as endogenous and fusion protein recruitment as well as affinity tagging [76]. Both dCas9 tethering and CRISPR-Disp can be used to elucidate the functions of cis-acting lncRNAs.

3.3. Determining potential domains within a lncRNA

Determining the structure of a lncRNA can elucidate its potential functions and aid in gRNA design. Many lncRNAs have complex secondary and tertiary structures that provide interfaces for protein, RNA, or DNA interactions [78]. Unlike many protein-coding genes, many lncRNAs are low in abundance [79] and poorly conserved [80]. This poses challenges to the identification of functional domains within lncRNAs. Furthering complicating matters, RNA-binding proteins may bind to small, conserved regions along a lncRNA transcript, making it difficult to distinguish whether the lncRNA or the protein are mediating the observed phenotypes. CRISPR-Disp can be used to analyse these domains. Using this system, uncharacterized lncRNA domains can be targeted to loci of interest to investigate their potential roles. Tiling CRISPR can also be used to examine functional lncRNA domains. Wang et al. (2019) developed this novel CRISPR/Cas9 approach to characterize the functional domains of Xist, a lncRNA involved in X-chromosome inactivation [81,82]. By introducing over 1,500 gRNAs, tiling the entire Xist transgene, the authors probed for clustered mutations from overlapping gRNAs, indicating a functional domain and successfully identifying essential regions of the gene [81]. Screens that utilize the Tiling CRISPR technology will have the potential to characterize functional domains of lncRNAs.

3.4. In vivo studies

One of the most comprehensive methods of determining the function of a lncRNA is studying its role in animal models. Traditional in vivo studies use the Cre-LoxP system, in which tissue-specific Cre recombinase (Cre) performs targeted deletions of genomic DNA between two LoxP sites [83]. Although this system does provide temporal and tissue-specific control, it can be a costly and time-consuming process [84]. The CRISPR/Cas9 system offers an alternate method of genetic modification in organisms. As previously described, this has been achieved using pairs of gRNAs to precisely delete up to 23kb of a lncRNA locus in a murine model [17]. Silencing lncRNA expression has also been performed by inserting a polyA cassette within an exon via the CRISPR/Cas9 system to promote cleavage of the transcript and terminate transcription [85]. Additionally, CRISPRa has been developed to activate lncRNA loci in vivo using a combination of the piggyBac transposon and Cre-inducible dCas9 systems [86].

Non-specific gRNA targets must be taken into account when creating in vivo systems to study lncRNAs [87]. When whole genome sequencing was performed on mice edited with the CRISPR/Cas9 system, a surprising number of single nucleotide variants were identified [87]. Although the system can induce significant off-target mutations, a recent study developed a method that provides verification of in vivo off-targets (VIVO) [88]. The VIVO workflow consists of two steps: in vitro reporting of cleavage effects by sequencing to identify potential off-target cleavage sites and off-target mutation validation in the CRISPR/Cas9-edited target tissues by deep sequencing [88]. This method can be combined with in silico techniques, which design gRNAs with the lowest number of similar genomic sites, to assess the accuracy of gRNAs [88]. This emerging technology shows promise for our future ability to perform functional studies of lncRNAs in animal models by minimizing potential off-target effects.

3.5. CRISPR library screens

CRISPR library screens introduce a pool of gRNAs into a cell population, knocking out (as occurs in the traditional case of Cas9) various genes in order to identify how genes may be enriched or depleted in response to a selective pressure. CRISPR library screens can be done in vitro and in vivo, using paired gRNAs to knockout lncRNAs or single gRNAs to target splice sites [19,32,89,90]. Screens have also utilized both the CRISPRi and CRISPRa systems [89,91]. These studies offer opportunities to identify novel lncRNA roles and interactions in an unbiased and genome-wide scale, elucidating both cytoplasmic and nuclear interactions. However, genome-wide lncRNA screens can be difficult due to the size of lncRNAs and inability of point mutations to yield the same inactivations as screens for protein-coding genes.

CONCLUSIONS

Despite low expression [79] and poor conservation [80], the CRISPR/Cas system provides innovative ways to investigate the potential roles of lncRNAs through knockout, knockdown, overexpression, and imaging approaches (Fig. 1). In addition to many of the techniques mentioned in this review, various other advancements are posed to greatly increase our knowledge of both RNAs and lncRNAs. The catalytically inactive CasRx (dCasRx) system has been employed in the manipulation of RNA splicing [15]. Because lncRNAs undergo splicing similar to mRNAs [92], this system can be used to perform splice isoform engineering on these transcripts. Additionally, dCas13 has been shown to effectively edit RNA when fused with adenosine deaminase acting on RNA type 2 (ADAR2) enzymes [63]. This system, termed RNA Editing for Programmable A to I Replacement (REPAIR), is not limited by sequence restrictions and has the potential to support lncRNA editing. A similar system entitled RESCUE (RNA Editing for Specific C-to-U Exchange) expands upon the REPAIR system, allowing for RNA editing of A to I and C to U [93]. The CRISPR/Cas13 systems have also been engineered to perform sensitive RNA detection through methods such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) [94]. These detection methods can likely be utilized in lncRNA studies due to the programmable nature of the system. Gao et. al (2021) were able to manipulate the CRISPR system to reliably quantify low-copy-number lncRNA expression levels using an endogenous transcription-gated switch with a CRISPR-activator-associated reporter [95]. The CRISPR/Cas9 technology may also be useful in manipulating the epigenetic regulation of lncRNA genes by utilizing dCas9 proteins fused with the catalytic domain of acetyltransferase [96]. This would allow for the controlled transactivation of various lncRNA loci, thus providing another systematic method to interrogate lncRNA functions.

Figure 1.

CRISPR uses in studies of lncRNAs. The CRISPR/Cas9 technology can be used to target various regions within a lncRNA gene, manipulating the lncRNA expression to gain functional insights. The CRISPR/Cas9 technology can be used to produce knock-in phenotypes (1,2), inserting a polyA stop cassette downstream of the transcription start site to decrease transcription or to encode a MS2 tag, as used in the CERTIS system, to localize lncRNAs. Additionally, the Cas9-gRNA complex can be directed to the beginning and end of the locus (3), splice sites (5), as well as transcription factor binding sites within the promoter region (4). These techniques will likely result in the partial or complete reduction of lncRNA levels within a cell. CRISPR activation, CRISPRa (7), and CRISPR interference, CRISPRi (6), can be used to overexpress or silence lncRNA expression via catalytically dead Cas9 (dCas9) without permanent genomic editing. CRISPRa employs a dCas9-gRNA complex fused with transcription activators (i.e. VP64, p65, and Rta) to robustly activate the transcription of the lncRNA gene. Conversely, the transcription of a lncRNA gene can be inhibited by CRISPRi, which utilizes a dCas9-gRNA complex fused with KRAB, a transcription repressor. Various orthologs of Cas13 can be utilized in lncRNA knockdown (8) in which a gRNA targets catalytically active Cas13 to single-stranded lncRNAs, leading to degradation of the molecule. Cas13 can also be used to visualize lncRNAs (9) by fusing dCas13 to a fluorescent protein

In addition to these incredible opportunities the CRISPR system offers to further our understanding of lncRNAs, with futher research and development, the CRISPR system could theoretically be clinically utilized to manipulate lncRNAs in a therapeutic context. CRISPR has corrected point mutations in vivo in mice and has been used ex vivo in the treatment of cancers, suggesting that CRISPR could be used to treat disease in vivo in the future [97–99]. Because lncRNAs are lowly-expressed and often tissue- and cell-specific, they could be good options as therapeutic targets. The major concern when considering CRISPR use in patients is off-target effects, though this may be less of an issue when targeting lncRNA compared to protein-coding genes. In addition to this serious concern, studying in vivo targeting of lncRNAs is complex due to the lack of conservation between species. Lastly, the prospect of using CRISPR to target lncRNAs therapeutically does present ethical concerns that need to be addressed prior to clinical use of this technology. From manipulating the traditional Cas9 knockout to utilizing inventive approaches such as CRISPR-Disp, the CRISPR/Cas system provides endless opportunities for innovative studies regarding the function on lncRNAs.

Funding Statement

This work was supported by the National Cancer Institute [ZIA BC 011646].

Disclosure statement

No potential conflict of interest was reported by the authors.