Neville E. Sanjana's team at New York University published a research paper titled "Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells" in the international academic journal Cell. The study developed a transcriptome-scale CRISPR screening technology based on CRISPR-Cas13 targeting RNA, and used this technology to screen and identify 778 essential lncRNAs in 5 human cells from different tissues, indicating that many lncRNAs are not junk, but play an essential and important role in human cancer and development.
This study established a powerful screening tool that can be used to systematically study the functions of non-coding transcripts and pave the way for identifying lncRNAs that have functions in any phenotype or disease. In addition, the screening tool has a wide range of applicability and is not limited to lncRNAs. It can also be directly applied to the screening of other non-coding RNAs, including enhancer RNAs and circular RNAs.
The advent of CRISPR gene editing technology has revolutionized biomedical research. Most CRISPR gene editing uses the Cas9 enzyme to edit genes at the DNA level. In recent years, Cas9-based CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have been used to screen and identify functional lncRNAs. Although these technologies are valuable, they often encounter unexpected on-target activity, that is, binding at the expected genomic site but interfering with other nearby genes. In addition, perturbing lncRNA sites at the DNA level may also inhibit functional DNA elements unrelated to lncRNA transcripts, resulting in distorted screening results.
Compared with Cas9 enzymes, Cas13 enzymes target and edit RNA without disrupting nearby protein-coding genes and other DNA regulatory elements. To overcome the limitations of current CRISPR screening for functional lncRNAs, the research team developed an RNA-targeted CRISPR screening method based on CRISPR-Cas13. It can systematically interfere with lncRNAs with transcript and strand specificity at the transcriptome level to ensure that no unexpected regulation of nearby genes or functional DNA elements occurs within the site.
The research team used massively parallel CRISPR-Cas13 forward transcriptome screening of 6199 lncRNAs in five different human cell lines (HAP1, HEK293T, K562, MDA-MB-231, THP1), from which a set of shared core essential lncRNAs (affecting cell survival, proliferation or differentiation) were identified. They were compared with nearby protein-coding genes in terms of their essentiality, analyzed transcriptome changes after perturbations in single cells, and described their key roles in development and cancer progression.
Using lncRNA expression profiles from 7 organs and 26 developmental stages (from 4 weeks after conception to old age), the research team designed a CRISPR-Cas13 library with the goal of targeting all lncRNAs expressed at 5 reads per kilobase of transcript per million mapped reads (RPKM) or above in at least one organ or donor at all stages. The research team also targeted an additional 2500 lncRNAs from recent screenings using DNA-targeted CRISPR and lncRNAs present in the lncRNA database (lncRNAdb). The CRISPR-Cas13 library designed by the research team contains 75,000 gRNAs for targeting 6,199 lncRNAs and 4,390 nearby protein-coding genes.
Screening results of five different human cell lines (HAP1, HEK293T, K562, MDA-MB-231, THP1) showed a total of 778 essential lncRNAs that are essential in at least one cell line.
Figure 1. Transcriptome-scale RNA-targeting CRISPR screens to identify essential lncRNAs in human cells. (Liang W W, et al., 2024)
Further analysis showed that of the 778 essential lncRNAs, 61% (477) were cell type specific (that is, essential in only one cell line), 33% (255) were partially shared (essential in 2-4 cell lines), and only 6% (46) were essential in all 5 cell lines. Next, the research team further studied the 46 shared essential lncRNAs in detail. These included MALAT1 and MIR17HG, the former is a ubiquitous lncRNA that has been shown to regulate cell motility and cancer metastasis; the latter is a miRNA host gene that promotes cancer progression. Only 6 of the 46 essential lncRNAs were found in previous studies of essential lncRNAs, and they were usually found in only one cell line.
The research team further analyzed whether these essential lncRNAs would regulate protein-coding genes nearby, a mechanistic question that has never been studied in non-coding RNAs. The study found that the vast majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using single-cell transcriptome analysis, the research team also found that the loss of these essential lncRNAs impairs cell cycle progression and drives apoptosis. Many essential lncRNAs show dynamic expression across tissues during development, with high expression in tissues early in human development and low expression in later stages, indicating that some lncRNAs play an important role in human development.
The research team also analyzed approximately 9,000 primary tumors and found that lncRNA expression was altered in specific types of tumors. And further identified those lncRNAs whose expression in tumors was associated with better or worse cancer survival, thus providing new biomarkers and potential therapeutic targets.
Figure 2. Essential lncRNAs are differentially expressed in tumors and correlate with survival. (Liang W W, et al., 2024)
Overall, this transcriptome-scale functional lncRNA screen advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screening using CRISPR-Cas13.
Reference
Liang W W, et al. Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells. Cell, 2024.