TABLE 2.
Current applications of Cas13d in basic research and biomedical therapeutics.
| Study summary | Organism used | Efficiency | Gene involved | References |
|---|---|---|---|---|
| CRISPR-RfxCas13d system has been implemented on several model organisms to study gene function and developmental changes in different organisms. This study demonstrated that CRISPR-RfxCas13d can be utilized as an efficient knockdown tool to investigate the maternal and zygotic gene function in various animal embryos. | Zebrafish | 76% | tbxta, szrd1, dnd1, smad5, alk8, oep, smad2, NANOG, brd3a, brd3b, and brd4 | Kushawah et al. (2020) |
| Medaka | rx3 | |||
| Killifish | firstly, mRNAs encoding gfp, rfp, mCas13d were introduced, three gRNAs targeting rfp were introduced as well. | |||
| Mouse | ubtf and emg1 | |||
| To develop a programmable platform for RNA targeting of known phenotypic genes using genetically encoded programmable RNA-targeting RfxCas13d (CasRx) system have shown limitation like unexpected toxicity and lethality of endogenously in Drosophila melanogaster | Drosophila | NK | Ubiq, NOTCH gene, White gene, Y (Yellow) gene | Buchman et al. (2020) |
| CRISPR-CasRx system was incorporated in glial cells, in vivo and targeted the single RNA-binding protein, polypyrimidine tract-binding protein 1 for the conversion of glia to neuron using CasRx -mediated knockdown of Ptbp1 shows a potential therapeutic approach for tackling neurodegenerative diseases and brain damage due to neuronal loss. | Mouse | 76–87% | Ptbp1 | Zhou et al. (2020) |
| dRfxCas13d was incorporated into the neuronal model of frontotemporal dementia cells to maneuver pathological alternative splicing of tau pre-mRNA which result in successful alleviation of dysregulated tau isoform. | Patient-derived human-induced pluripotent stem cells (hiPSCs) | >90% | MAPT | Konermann et al. (2018) |
| Cas13d nuclease activity was able to efficiently knock-down various exogenous and endogenous genes which are involved in apoptosis, gene amplification, metabolism, and glycosylation. | Chinese hamster ovary (CHO) cell | 80–90% | GS, BAK, BAX, PDK1, and FUT8 | Shen et al. (2020) |
| Targeting metabolic genes in mouse hepatocytes by active RfxCas13d, clearly presented to be an efficient strategy to carry-out regulatory knockdown of metabolic genes that can be efficiently used in the treatment of metabolic diseases. | mouse | 16–61.6% | Pten, Pcsk9, and lncLstr | He et al. (2020) |
| CasRx is able to knock down the transcript of mutant KrasG12D and further abolishes the irregular activation of downstream signaling pathways resulting in suppression of tumor growth. | AsPC-1, PANC-1, MIAPaCa-2, and H6c7 Pancreatic cancer cell line and nude mice. | 50% | Mutant KrasG12D | Jiang et al. (2020) |
| Silencing of lncRNA MIR497HG via CRISPR/Cas13d induced bladder cancer progression through promoting the crosstalk between Hippo/Yap and TGF-β/Smad signaling | Human bladder cancer cell lines T24, 5637, RT4, UM-UC-3, SW780, and TCCSUP. | NK | MIR497HG | Zhuang et al. (2020) |
| Aptazyme mediated CRISPR/Cas13d gene-editing system can efficiently sense hTERT and selectively inhibits the progression of bladder cancer cells | bladder cancer cell line 5,637 and T24 | NK | hTERT | Zhuang et al. (2021) |
| CasRx system successfully constructed an inducible expression system and applied it for efficiently repressing the expression of a green fluorescent protein (GFP) in E. coli | E. coli | ∼70% at mRNA level and 30–50% at the protein level | green fluorescent protein (GFP) | Zhang et al. (2020) |
| A new tool, the CRISPR-based RNA-United Interacting System (CRUIS) was developed, which captures RNA–protein interactions in living cells by combining the power of CRISPR while fused to proximity enzyme PafA. CRUIS was able to show a similar interactome profile of NORAD (Noncoding RNA activated by DNA damage) and CLIP (crosslinking and immunoprecipitation) based methods. | HEK239T cell line | 20–70% | CXCR4, p21, NORAD | Zhang et al. (2020) |
| CRISPR-Cas13 systems were used to deliver APEX2 to the human telomerase RNA hTR with high specificity enabling RNA interactome profiling on a 1-min time scale. ALKBH5 is able to erase the m6A modification on endogenous hTR. | NK | hTR, APEX2, ALKBH5 | Han et al. (2020) | |
| Xie et al., found that REMOVER (reengineered m 1 A modification valid eraser), a Cas13d based CRISPR tool which specifically demethylated the m1A of MALAT1 and PRUNE1 RNAs and also noted that it significantly increased their stability. Thus, their studies have established that REMOVER can be used as a tool for targeted RNA demethylation of specific m1A-modified transcripts. | HEK239T cell line | NK | MALAT1 and PRUNE1 | Xie et al. (2021) |
| RfxCas13d coupled with fluorescent-labeled crRNA along with dCas9-fluorescent crRNA system has been used for real-time simultaneous visualization of transcript RNA and genomic DNA in the method known as CRISPR LiveFISH (live-cell fluorescent in situ hybridization) | Human osteosarcoma cell line U2OS cell line | NK | PPP1R2, SPACA7 | Wang et al. (2019) |
| RfxCas13d did not exert collateral cleavage effect in plants and was able to efficiently target two RNA viruses in parallel when crRNAs targeting two viruses were expressed in tested plants. CasRx showed robust interference in both transient and stable overexpression assays when compared to the other Cas13 variants tested. | Nicotiana benthamiana | NK | Replicase gene of tobacco mosaic virus (TMV) and tobacco rattle virus (TRV) | Mahas et al. (2019) |
| RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. | HEK293T derived Lenti-XTM cells and TZM-bl cell line | >90% | gRNAs having active site of the HIV protease enzyme (BR23), the central polypurine tract (BR29), catalytic core domain of integrase (BR34), and the c-terminal domain (BR04) | Nguyen et al. (2021) |
| The RfxCas13d based PAC-MAN (prophylactic antiviral CRISPR in human cells) was shown to efficiently cleave SARS-CoV-2 RNA fragments and inhibit the influenza A virus. | A549 and MDCK cells | NK | RdRP or N gene regions of SARS CoV 2 and eight negative-sense RNA segments of IAV including RNA polymerase subunits, hemagglutinin, Nucleoprotein, Neuraminidase, M1, M2, NS1, and NEP | Abbott et al. (2020) |
| Two Cas13d orthologs were introduced for detection of low variant allele fraction, 0.1% T790M. Overall, this study demonstrated that both EsCas13d and RspCas13d could robustly detect target RNA carrying special single-nucleotide variation with high specificity and sensitivity. | E. coli BL21 (DE3) | 72% | EGFR | Qiao et al. (2021) |
NK-signifies that the efficiency of knockdown or targeting efficiency of Cas13d is not known.