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PubMed2025

A quick guide to evaluating prime editing efficiency in mammalian cells.

According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.

Gene Editing↗
PubMed2024

Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection.

CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting.

CRISPR-Cas Systems↗
PubMed2002

RNA and RNA-protein complexes as targets for therapeutic intervention.

Today, the majority of pharmaceuticals developed to treat cancers and viral/bacterial infections target cellular, bacterial or viral proteins known to be associated with a given pathology. Although proteins are the focus of most current drug discovery efforts, exciting new research has recently begun which aims to exploit ribonucleic acid (RNA) and RNP particles as novel targets for pharmaceutical development. These RNA-targeted research efforts have been fueled by an increased appreciation for the central role played by RNA and RNA-protein interactions in many biological processes and diseases, together with a better understanding of RNA structure and an improvement in biophysical/biochemical techniques available to study RNA. As for protein targets, genome sequencing is greatly accelerating the identification of human and microbial RNA transcripts for targeted drug discovery. With this explosion in the number of potential RNA and RNP targets, the effective development of specific small molecule RNA-based drugs requires robust and general approaches for detecting and quantifying RNA-ligand interactions, which can be used as high-throughput screens (HTS) and for obtaining rapid structural information to guide rational drug design. In this review, an overview of the potential for therapeutic intervention based on RNA and RNP targets is presented, together with recent efforts to develop generally useful nuclear magnetic resonance (NMR) and fluorescence binding assays for screening and optimizing drugs aimed at RNA and RNP targets.

Animals↗
PubMed2002

The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: identification of SBP2 amino acids important to SECIS RNA binding.

Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3' untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.

Amino Acid Sequence↗
PubMed2002

Regulation of bacterial RNase P ribozyme reaction by divalent cation and guide DNA.

The RNA subunit of bacterial ribonuclease P (RNase P) is a ribozyme which can cleave a canonical cloverleaf tRNA precursor and a hairpin RNA with a CCA-3' tag sequence as its substrate. With high concentration of Mg ion, the ribozyme as well as holo enzyme internally cleaves certain tRNAs in vitro. We denoted this unusual reaction as hyperprocessing. By controlling magnesium ion concentration for the reaction and also by forcing the RNA shape with external guide DNAs, we could regulate the hyperprocessing reaction by the bacterial RNase P enzymes. These techniques will lead the RNase P ribozyme to more designable and more applicable RNA-cleaving enzyme.

Bacteria↗
PubMed2025

Lentiviral CRISPRa/i in the adult prairie vole brain: modulating neuronal gene expression without DNA cleavage.

Prairie voles (Microtus ochrogaster) are a powerful model for studying the neurobiology of social bonding, yet tools for region- and cell type-specific gene regulation remain underdeveloped in this species. Here, we present a lentivirus-mediated CRISPR activation and interference (CRISPRa/i) platform for somatic gene modulation in the prairie vole brain. This system enables non-mutagenic, titratable regulation of gene expression in the adult brain without germline modification. Our dual-vector system includes one construct expressing dCas9-VPR (VP64-p65-Rta) referred to as CRISPRa or dCas9-KRAB-MeCP2 (Kruppel-associated box-methyl CpG binding protein 2), referred to as CRISPRi under a neuron-specific promoter, and a second construct delivering a U6-driven sgRNA (single guide RNA) alongside an elongation factor 1 alpha (EF1α)-driven mCherry reporter. We detail the design, production, and stereotaxic delivery of these tools and demonstrate their application by targeting four genes implicated in social behavior (Oxtr, Avpr1a, Drd1, and Drd2) across two mesolimbic brain regions: the nucleus accumbens and ventral pallidum. Gene expression analyses confirmed robust, bidirectional transcriptional modulation for selected targets, establishing a proof of concept for CRISPRa/i in this non-traditional model. The dual-vector design is readily adaptable to other gene targets, cell types, and brain regions, and can be multiplexed to provide a flexible and scalable framework for investigating gene function in behaviorally relevant circuits. These advances represent the first successful implementation of somatic CRISPRa/i in prairie voles and expand the genetic toolkit available for this species.

Avpr1a↗
PubMed2026

Extracellular vesicles-mediated delivery of SpCas9 RNPs for therapeutic gene editing in Spinocerebellar Ataxia Type 3.

Spinocerebellar Ataxia Type 3 (SCA3) is a neurodegenerative dominantly-inherited disorder caused by an overexpansion of a CAG tract within the ATXN3 gene, conferring toxic properties to the ataxin-3 protein. Genome editing with CRISPR-Cas9 enzymes is a promising strategy to inactivate mutant ATXN3 alleles, however, in vivo delivery remains challenging. Extracellular vesicles (EVs) are promising delivery vehicles for Cas9 and single guide RNA (sgRNA) ribonucleoproteins that minimize genomic exposure to highly active endonucleases. In this study, we designed SpCas9 with a palmitoylation motif that enables SpCas9 and sgRNA enrichment into EVs. Introduction of a photocleavable linker - PhoCl - allowed the photo-inducible release of SpCas9 from the palmitoylation motif in EVs, increasing target engagement to ATXN3 in vitro. EVs loaded with SpCas9 ribonucleoproteins resulted in ATXN3 knockout in SCA3 patient-derived iPSCs and two SCA3 animal models. These findings highlight an innovative route for transient delivery of gene editing tools. This approach provides a promising therapeutic platform for the treatment of genetic diseases, including SCA3.

Humans↗
PubMed2002

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

Animals↗
PubMed2026

When Homing Endonuclease Meets Transposon: The OMEGA System.

Sequence-specific DNA endonucleases have made significant contributions to biology, biotechnology, and medicine; restriction enzymes and homing endonucleases are among classic examples. The demonstration of programmable genome editing using Cas9 in the CRISPR-Cas system, in which the target DNA sequence is recognized by base pairing with a guide RNA, revolutionized the field of genome engineering, making target selection more flexible and convenient. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is an RNA-guided DNA endonuclease composed of a TnpB, IscB, IsrB, or Fanzor protein, and a structural RNA designated reRNA or ωRNA. The OMEGA system is present in the three domains of life as an auxiliary component of transposons. The OMEGA system cuts DNA in an allele from which a transposon is excised and triggers recombination to reinstate the transposon at the same position. This "transposon restorative homing" redefines the OMEGA system as a homing endonuclease. In this review, the selfish aspects of the OMEGA system are discussed in the historical context of homing endonuclease research.

Cas12↗
PubMed2001

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease.

We examined the suppression effect of HIV-1 expression by cleavage of the HIV-1 RNA gene, using the catalytic RNA subunit RNase P and the 3'-half tRNA [External Guide Sequence (EGS)] in vivo. The vectors were designed to express an anti-HIV EGS, U5, which targets the 5' leader sequence. We constructed an EGS expression vector, that used the tRNA(met) or U6 promoter as an expression cassette for EGS. RNase P cleaves the targeted HIV-1 mRNA when they are in a complex with the EGS. To test the antiviral efficacy of these EGS vectors, we have cotransfected into COS cells with the HIV-1 proviral DNA (pNL4-3 Luc) and the plasmid expressing the EGS from the tRNA(met) or U6 promoter. HIV-1 expression was inhibited by the tRNA-EGS-1 and U6-EGS-1 from the tRNA(met) and U6 promoters, respectively. No difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters could be detected.

Animals↗
PubMed2001

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Animals↗
PubMed1993

Ligation-based DNA diagnostics.

A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.

DNA↗
PubMed2026

Rational and computation-assisted engineering of a compact and efficient CRISPR-Cas12f genome editor.

The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. Single guide RNA (sgRNA) scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.

CRISPR-Cas12f↗
PubMed2002

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Base Sequence↗
PubMed1992

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.

Base Sequence↗
PubMed2003

Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

Anemia, Sickle Cell↗
PubMed2026

Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.

CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.

Solanum lycopersicum↗
PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗