You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest of our genome could possibly be made up of. The answer is… drum roll please… non-coding RNAs! You probably didn’t see that coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important roles in many cellular and regulatory processes, but much of their functionality is yet to be unveiled as they are more difficult to functionally manipulate and study in comparison to protein-coding genes (Rinn and Chang 2012).
Scientists have determined the function of many protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe the effects. Luckily for us, CRISPR is an incredibly versatile system that can be tweaked to target ncRNA.
CRISPR/Cas9 techniques and targeting methods
Knock-out: Targeting short vs. long ncRNAs
The CRISPR/Cas9 system is often used to knock-out, or permanently disable, protein-coding genes by generating insertions or deletions in the DNA sequence. This same approach is effective to target short ncRNAs, such as microRNAs that are around 20 base pairs long. Using a single guide RNA (sgRNA) to create a double-strand break works well because very small deletions and insertions in the DNA sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).
Simple knock-out experiments are trickier when it comes to long non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so small deletions or insertions may not result in a functional loss (Ho et al. 2014). To overcome this, paired gRNAs can be used to target specific sizes of a DNA fragment and turn off the function by removing the full ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for targeting thousands of long ncRNAs at the same time (Liu et al. 2018). However, removing large lncRNA fragments with multiple gRNAs poses the risk of disrupting adjacent genes, especially those regulated by or overlapping with the lncRNA (Basak and Nithin 2015, Horlbeck et al. 2021).
Alternatively, off-target effects can be minimized by disrupting lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting to cut out the majority of the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters to knock out lncRNA function (Hazan and Bester 2021; Zibitt et al. 2021). Mutating splice sites is particularly effective as it can result in the retention of introns or the removal of exons. Disrupting promoter or transcription start site function would also help to reveal the impacts of a lncRNA on nearby genes and its role in gene expression, though this may still unexpectedly affect nearby genes (Figure 1A-C).
Find CRISPR plasmids at Addgene!
Knock-in: Introducing tags or stop signals
Once Cas9 generates double-strand breaks, a new DNA sequence can be knocked-in, or introduced, into the break sites. Knock-in experiments are important for visualizing ncRNA localization and function, but directly incorporating very long ncRNAs is impractical. Instead, tags or stop elements can be inserted to disrupt the transcription of the ncRNA (Hazan and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure 1D). While this method can be time-consuming, it is relatively effective in reducing off-target effects since promoter and enhancer sequences remain intact (Engreitz et al. 2019). The interactions between ncRNAs and adjacent genes are complex and not yet fully understood, so it is important to carefully select the target locations of knock-in studies to avoid off-target effects (Zibitt et al. 2021).
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| Figure 1: A schematic illustration of CRISPR/Cas9-based approaches to study ncRNAs. (A) Transcription start site (TSS) deletion. (B) Mutations of splice sites. (C) Removal of an exon or a large genomic fragment. (D) Knock-in: insertion of a synthetic polyadenylation (spA) signal. Figure and caption adapted from Hazan and Bester 2021. |
Alternative CRISPR systems for targeting ncRNAs
While CRISPR/Cas9 has proven to be an extremely useful system for modifying and assessing ncRNA function, it may fall short when it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not effective for your experiments, there are plenty of other CRISPR-based options that can target ncRNAs.
CRISPRi and CRISPRa
For scientists who want to modulate ncRNA expression without permanently altering the DNA, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily block the transcription machinery of a target gene or DNA that codes for ncRNA. CRISPRa, on the other hand, recruits transcriptional activators such as VP64, SAM, or SunTag to specific ncRNA loci to enhance or upregulate ncRNA expression. This approach is useful for lncRNAs because they regulate nearby genes and interact with specific chromatin structures or other local genomic elements (Hazan and Bester 2021).
Other Cas variants
Another option is CRISPR/Cas13, which targets RNA instead of DNA and selectively degrades RNA molecules. This system is often used to knock down lncRNAs and has fewer off-target effects. Newer Cas protein variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave single-stranded RNA using gRNAs to specifically edit or degrade targeted lncRNAs. The Cas14 variant is a smaller and more versatile option. It can be used to target lncRNAs with high specificity, particularly in applications requiring smaller delivery systems (Zibitt et al. 2021).
Even though it is more difficult to study ncRNA, CRISPR systems offer a number of different options. By carefully considering your ncRNA of interest, you should be able to devise an experiment that can tell you more about its function.
Resources and references
Resources on addgene.org
More resources on the Addgene blog
Targeting RNA with Cas13a (C2c2)
Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering
References
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337(6096):816-821. doi:10.1126/science.1225829
Hazan J, Bester AC. CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021;7(4):79. doi:10.3390/ncrna7040079
Horlbeck MA, Liu SJ, Chang HY, Lim DA, Weissman JS. Fitness effects of CRISPR/Cas9-targeting of long non-coding RNA genes. Nature biotechnology. 2020;38(5):573. doi:10.1038/s41587-020-0428-0
Rinn JL, Chang HY. Genome Regulation by Long Noncoding RNAs. Annual Review of Biochemistry. 2012;81(Volume 81, 2012):145-166. doi:10.1146/annurev-biochem-051410-092902
Liu Y, Cao Z, Wang Y, et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol. Published online November 5, 2018. doi:10.1038/nbt.4283
Zibitt MS, Hartford CCR, Lal A. Interrogating lncRNA functions via CRISPR/Cas systems. RNA Biology. 2021;18(12):2097. doi:10.1080/15476286.2021.1899500
Engreitz JM, Haines JE, Perez EM, et al. Local regulation of gene expression by lncRNA promoters, transcription, and splicing. Nature. 2016;539(7629):452. doi:10.1038/nature20149
Jiang Q, Meng X, Meng L, et al. Small indels induced by CRISPR/Cas9 in the 5’ region of microRNA lead to its depletion and Drosha processing retardance. RNA Biol. 2014;11(10):1243-1249. doi:10.1080/15476286.2014.996067
Basak J, Nithin C. Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front Plant Sci. 2015;6. doi:10.3389/fpls.2015.01001
Ho TT, Zhou N, Huang J, et al. Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Research. 2014;43(3):e17. doi:10.1093/nar/gku1198
Topics: CRISPR, CRISPR 101
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