CRISPR-Cas13: a powerful tool for studying lncRNAs
Figure 1 – Transcriptome scale CRISPR-Cas13 screen to find essential lncRNAs
Abstract
The human genome is pervasively transcribed into RNA and encodes thousands of long noncoding RNAs (lncRNAs), of which less than 1% have a clear function. Recently, CRISPR-Cas9 pooled screens using CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have been applied to identify functional lncRNAs. However, they often suffer from unintended on-target activity by perturbing additional nearby genes. In this research, the authors perturbed 6,199 genes using a massive transcriptome-scale CRISPR-Cas13 screening, avoiding potential on-target activity of Cas9-based ones, to find essential lncRNAs in human cells.
Review
LncRNAs are present in all mammalian cells. These RNAs are at least 200 nucleotides long, get spliced and polyadenylated just like normal mRNA, but are not transcribed into protein. Studying lncRNAs is challenging due to their low sequence conservation, low abundance and tissue-specific expression. For these reasons, just 1% are linked to a clear function, often regulatory. Previous researchers used CRISPR-Cas9-based technology to study lncRNA functions, acting at genome-scale. Unfortunately, these approaches suffer from unintended on-target activity, by perturbing additional nearby genes.
To overcome the limitations of CRISPR-Cas9-based technologies, the researchers Wen-Wei Liang, Simon Müller et al. developed RNA-targeting CRISPR-Cas13 screens to systematically perturb lncRNAs at the transcriptome scale1. With this technique, they perturbed 6,199 lncRNAs in five distinct cell lines, identifying a core set of essential human lncRNAs; compared their essentiality to nearby protein-coding genes; investigated the change in transcriptomic profile upon lncRNAs perturbation at single-cell resolution; and described key roles in development and cancer progression. This research yields new putative cancer biomarkers and opens doors to new potential therapeutic targets.
Transcriptome-wide CRISPR-Cas13 screens to identify essential human lncRNAs
The authors made a library of ~70,000 guide RNAs (gRNAs) to target 6,199 lncRNAs and 4,390 protein coding genes (PCGs) with ~8 gRNAs each. They used five cell lines: HAP1 (Haploid cell line 1), HEK293FT (Human Embryonic Kidney), K562 (hematopoietic origin), MDA-MB-231 (from breast carcinoma) and THP-1 (human monocytic cell line). The researchers engineered these cell lines to express a nuclear-localized RfxCas13d effector under doxycycline-inducible control and infected them with lentiviral gRNAs (low MOI). Cas13 expression was induced via doxycycline addition. Finally, they harvested genomic DNA from this cell at 0, 7, and 14 days post-Cas13 infection, and computed the change in gRNAs abundances with amplicon sequencing. By looking at depleted gRNAs, they found 778 essential lncRNAs. For the PCGs, the results obtained were comparable to the previous studies that used Cas9-based approaches. They also found that, among those essential lncRNAs, 61% (477) are cell-type-specific, 33% (225) are partially shared and 6% (46) are shared across cell types. Compared with PCGs, essential lncRNAs were more cell-type-specific, with less overlap between cell lines. As a reference, just 6 out of 46 shared essential lncRNAs were classified as essential in the previous studies.
To confirm their essentiality researchers performed three experiments: in the first one, they targeted 7 of the 46 shared essential lncRNAs with three independent gRNA and measured the cell growth via competitive cell growth assay. Next, they used the FUCCI system to demonstrate the perturbation of the cell cycle (e.g. MALAT1 depletion leads to accumulation of cells in G2-M phase). In the last experiment, researchers used annexin V staining to study cell apoptosis after depletion of essential lncRNAs, revealing that it consistently leads to an increase in the apoptotic cell population. This behavior varies in intensity depending on the depleted lncRNA.
Most essential lncRNAs operate independently of their nearest protein-coding genes
The researcher wondered whether the lncRNAs act independently from their nearby PCGs; with the transcriptome-wide CRISPR-Cas13 screen described above, they perturbed at the RNA level (so with great specificity) 5,452 pairs of lncRNAs and their nearest PCGs, in five different cell lines. The results showed that only a few lncRNA-PCG pairs were essential and, most often, only one between the lncRNA and PCG was essential.
To validate this observation, the authors chose three pairs of lncRNAs-PCGs, having either one essential gene (lncRNA or PCG) or both, and targeted them using three independent gRNA for each gene; next, they monitored the cell growth via a competitive growth assay, which confirmed the depletion only in essential genes found with the transcriptome-wide CRISPR-Cas13 screens. Moreover, one of the three lncRNAs examined, XLOC_020797, was classified as non-essential by the Cas13-based screen, while being classified as essential by a prior CRISPRi screen. This supports the idea that acting at the RNA level may avoid confounding on-target effects.
21 of the 43 shared essential lncRNAs have the paired PCGs also classified as essential in at least one cell line, and only 4 are essential in all cell lines; in 15 of the 21 shared essential lncRNAs-PCGs, the genes are very close (<1kb).
Given these results, the authors wondered if lncRNAs loci may contact PCGs in three-dimensional (3D) genome and found that, using a chromosome conformation capture (Hi-C) dataset, 53%–62% of essential lncRNAs are in the same topologically associating domains (TADs) of essential PCGs. To quantify direct 3D contact, they then performed H3K27ac Hi-ChIP in HAP1 cells and found that only a few lncRNAs loci (4%–11%) contact PCGs loci in 3D chromatin. Therefore, essential lncRNAs genes do not influence the essentiality of nearby PCGs. Next, researchers compared their set of essential lncRNAs to a CRISPRi-based set. Cas13-based screens found fewer essential lncRNAs, and those found only in CRISPRi-based screen are enriched for lncRNAs in close proximity (<1 kb) to a PCG, suggesting the inability of Cas9-based screens to target specific genes when the transcription start sites are close. In total, 725 essential lncRNAs not previously reported were found in this research.
Dynamic expression of essential lncRNAs during development and in specific tumors
The authors next analyzed the proliferation-associated pathways linked to lncRNAs.
To this aim, they designed a new Cas13 Library targeting 50 essential lncRNAs and 21 PCGs with 3 individual gRNA for each target (2 targeting the same gene and one barcode gRNA) and transduced both MDA-MB-231 and HAP1 cells with this library. Then they used the CaRPool-seq technique to couple pooled CRISPR-Cas13 perturbations with a single-cell transcriptomic readout, allowing the identification of the specific perturbations introduced in a single cell and their impact on the transcriptome.
They analyzed the expression patterns triggered by essential lncRNAs perturbations in both MDA-MB-231 and HAP1 cells. The results showed minimal impact on the expression of closest PCGs when the corresponding essential lncRNAs were perturbed. Interestingly, they observed a downregulation of the MYC and mTOR pathways. Additionally, perturbation of essential genes (lncRNAs and PCGs) consistently upregulates the tumor suppressor p53 pathway, while downregulating pathways associated with proliferation, such as mitotic spindle organization and cell-cycle checkpoints.
Then researchers wanted to assess if essential lncRNAs have distinct expression patterns in vivo. By using data from a recent developmental atlas, they found that essential lncRNAs show a widespread expression across different developmental stages and in different tissues, as opposed to non-essential lncRNAs. By analyzing essential lncRNAs expression at different developmental stages in the brain, heart, liver, and kidney, they found higher expression in early stages (where there is higher cell proliferation), and a decreased expression in later stages; instead, non-essential lncRNAs showed an opposite pattern of expression.
Since the identified essential lncRNAs are associated with proliferation, researchers hypothesized a correlation between these lncRNAs and cancer progression. To prove their hypothesis, they re-aligned RNA-seq data from The Cancer Genome Atlas (TCGA) to a reference lncRNAs transcriptome and found that essential lncRNAs are more often differentially expressed in tumors as compared with non-essential lncRNAs. To understand the functional role of lncRNAs, researchers identified, for each lncRNA, the PCGs with correlated expression patterns in TCGA primary tumors. They found that co-expressed PCGs of essential lncRNAs are enriched for cell proliferation, and that the enrichment is greatest for shared essential lncRNAs. Finally, researchers found that 36 out of 46 shared essential lncRNAs are associated with improved or worsen survival.
Conclusions
The increased specificity of gene targeting at the transcriptome scale, while avoiding confounding on-target effects – as seen with CRISPR-Ca9-based methods – highlights the power of CRISPR-Cas13-based methodologies to study complex and gene-dense genomic loci. However, this technique is broadly applicable and not limited to the study of lncRNAs. Moreover, this research links essential lncRNAs to development and cancer progression, discovering new transcriptomic biomarkers that in future may be used for cancer treatment and cancer screening. The increasing number of lncRNAs being found in deeper transcriptomic profiling has made a complete screen currently unfeasible. Future research should include a more extensive library and a broader range of cell types. Moreover, to assess the essentiality, the authors used only a single phenotypic readout – growth in cell culture – and measured it in only five cell lines. These are important limitations, considering the low abundance and high tissue-specificity of lncRNAs.
References
- Liang, Wen-Wei et al. “Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells.” Cell vol. 187,26 (2024): 7637-7654.e29. doi:10.1016/j.cell.2024.10.021