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At least 19 recordsLinked to original sources

PubMed2026

CRISPGen: A deep generative framework for multi-objective CRISPR/Cas9 guide RNA design via Conditional Latent Diffusion and Dual-Critic Reinforcement Learning.

MOTIVATION: The CRISPR-Cas9 system offers transformative potential for precision genome editing, yet its clinical translation remains constrained by the risk of unintended off-target double-strand breaks. While current discriminative models excel at evaluating pre-specified candidate guides, resolving the fundamental antagonism between on-target cleavage efficiency and off-target specificity within a fixed sequence search space remains a major challenge. RESULTS: We present CRISPGen, a unified deep generative framework that reframes sgRNA design as a multi-objective constrained sequence synthesis problem. It integrates (i) DNABERT-2 genomic-language embeddings, (ii) a conditional latent diffusion generator conditioned on a user-specified on-target efficiency target, and (iii) a dual-critic reinforcement-learning (RL) stage that couples a frozen on-target efficiency critic with a cross-attention off-target discriminator (validation Pearson R=0.8157) trained on a unified corpus of experimental off-target events from six detection platforms. Across 1000 generated sgRNAs, CRISPGen reduces the mean off-target discriminator score by 99.7% relative to the pre-RL baseline and, under an exhaustive whole-genome screen of all 302,631,056 NGG PAM sites in GRCh38, yields zero perfect-match and only 55 one-mismatch genomic hits. We further show, transparently, that the internal on-target critic saturates under RL optimization - an instance of Goodhart's Law - and therefore assess on-target viability using an independent external CRISPRon screen (mean 47.10/100). Repeating the RL fine-tuning stage under three random seeds (with the diffusion generator, DNABERT-2 embeddings, and off-target discriminator held fixed) yields a stable operating point across seeds. Full diversity, per-mismatch, and reproducibility statistics are reported in the Results. AVAILABILITY: Source code is available at https://github.com/malekpouri/CRISPGen; the pre-trained checkpoints and the 3,000,000-sequence library are hosted on Hugging Face (https://huggingface.co/malekpouri/CRISPGen-Checkpoints) and archived on Zenodo under DOI 10.5281/zenodo.21428641.

CRISPR-Cas9↗

PubMed2026

Structure and evolution-guided design of minimal RNA-guided nucleases.

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.

Humans↗

PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗

PubMed2025

Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.

Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.

Gene Editing↗

PubMed2025

Stacked mutations in multi-copy AHAS genes enhance sulfonylurea herbicide resistance in soybean.

Weeds are a major factor that negatively impact crop yields. Developing herbicide-resistant germlines is crucial for efficient weed control. Sulfonylurea- and pyrimidinyl benzoate-based herbicides inhibit the function of acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis of branched-chain amino acids in plants. To create soybean plants resistant to these classes of herbicides, we performed base editing of AHAS genes in Glycine max. A guide RNA was designed to target the codon for proline-182 in GmAHAS2, with the prediction that off-target base editing might also occur in the GmAHAS3 and GmAHAS4 genes. We selected six genome-edited soybean lines, each carrying distinct mutations in GmAHAS2, GmAHAS3, or GmAHAS4. These lines were treated with three different AHAS-targeting herbicides to evaluate resistance. The results show that the number of mutated GmAHAS genes and the mutation patterns significantly influence herbicide resistance.

Herbicide Resistance↗

PubMed2026

RNA structures regulate norovirus life cycle and enable rational attenuation in vivo.

Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell chemical probing, revealing conserved motifs that regulate viral function. Targeted disruption of specific structural elements reduces viral replication in cell culture, modulates translation in cis, and decreases viral RNA levels in animal infection models. These findings enabled the rational design of a genetically stable, attenuated virus that elicits protective immunity and limits viral replication upon secondary challenge. Together, this work uncovers essential roles for RNA structure in norovirus biology and establishes a generalizable framework for RNA structure-guided design of antiviral vaccines and therapeutics.

RNA structure↗

PubMed2005

Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency.

In RNA interference (RNAi), guide RNAs direct RNA-induced silencing complexes (RISC) to their mRNA targets, thus enabling the cleavage that leads to gene silencing. We describe a strong inverse correlation between the degree of guide-RNA secondary structure formation and gene silencing by small interfering (si)RNA. Unstructured guide strands mediate the strongest silencing whereas structures with base-paired ends are inactive. Thus, the availability of terminal nucleotides within guide structures determines the strength of silencing. A to G and C to U base exchanges, which involve wobble base-pairing with the target but preserve complementarity, turned inactive into active guide structures, thereby expanding the space of functional siRNAs. Previously observed base degenerations among mature micro (mi)RNAs together with the data presented here suggest a crucial role of the guide-RNA structures in miRNA action. The analysis of the effect of the secondary structures of guide-RNA sequences on RNAi efficiency provides a basis for better understanding RNA silencing pathways and improving the design of siRNAs.

Algorithms↗

PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗

PubMed2025

Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9 reveals the functional impact of allelic variation.

The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.

Candida albicans↗

PubMed2002

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences.

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequence-specific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4(+) T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4(+) T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4(+) cells expressing the RNase P-associated U5 EGS (560) molecule.

CD4-Positive T-Lymphocytes↗

PubMed2001

Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA.

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed 'external guide sequence' (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self-cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.

Animals↗

PubMed2025

Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.

Numerous high-specificity Cas9 variants have been engineered for precision genome editing. These variants typically harbor multiple mutations designed to alter the Cas9-single guide RNA (sgRNA)-DNA complex interactions for reduced off-target cleavage. By dissecting the contributions of individual mutations, we attempt to derive principles for designing high-specificity Cas9 variants. Here, we computationally modeled the specificity harnessing mutations of the widely used Cas9 isolated from Streptococcus pyogenes (SpCas9) and investigated their individual mutational effects. We quantified the mutational effects in terms of energy and contact changes by comparing the wild-type and mutant structures. We found that these mutations disrupt the protein-protein or protein-DNA contacts within the Cas9-sgRNA-DNA complex. We also identified additional impacted amino acid sites via energy changes that constitute the structural microenvironment encompassing the focal mutation, giving insights into how the mutations contribute to the high-specificity phenotype of SpCas9. Our method outlines a strategy to evaluate mutational effects that can facilitate rational design for Cas9 optimization.

Gene Editing↗

PubMed2002

An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes.

U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.

Animals↗

PubMed2026

CROP: a feature-independent context-aware method for CRISPR-Cas9 frameshift prediction.

MOTIVATION: The CRISPR-Cas9 complex has revolutionized genome-editing technologies. By designing a 20 nt-long guide RNA, a Cas9 nuclease can be guided to cleave almost any genomic target site (followed by NGG). The cleavage induces double-stranded DNA breaks, which are then repaired by cellular pathways. Accurate CRISPR-Cas9 repair-outcome prediction is essential for designing guide RNAs with desired genomic effects, such as gene knockout. A central challenge is quantifying the rate of frameshifts, i.e. repair-outcomes that lead to a change in the local length that is not a multiple of three. Previous methods for frameshift-rate prediction were trained on only a few experimental or cellular contexts, mostly relied on manually defined microhomology features, and were limited by sparse features and class labels. RESULTS: We developed CROP, a feature-independent context-aware repair-outcome prediction method. By aggregating specific repair outcomes as Δlength classes, CROP overcomes class sparsity. We designed CROP to work with variable input sequence lengths and output classes to utilize multiple datasets simultaneously. We benchmarked CROP against state-of-the-art repair-outcome prediction methods over 18 datasets, which we curated and standardized from various studies. Across all datasets, CROP outperformed all competing methods in frameshift-rate prediction. We performed cross-experiment and cross-cellular frameshift-rate predictions to investigate the generalizability of repair mechanisms. Finally, we show that CROP learned microhomology principles from raw sequences without explicit feature engineering, establishing an end-to-end architecture for CRISPR-Cas9 repair-outcome prediction that learns from multiple datasets. AVAILABILITY AND IMPLEMENTATION: CROP is available at https://github.com/OrensteinLab/CROP.

CRISPR-Cas Systems↗

PubMed2000

Tertiary core rearrangements in a tight binding transfer RNA aptamer.

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.

Amino Acyl-tRNA Synthetases↗

PubMed1996

Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides.

Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site--a complementary snoRNA is required for methylation at a specific site. Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when coexpressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.

Animals↗

PubMed2003

Characterization of the structure and variability of an internal region of hepatitis C virus RNA for M1 RNA guide sequence ribozyme targeting.

Accessibility to folded RNA and low potential of variation in the target RNA are crucial requirements for ribozyme therapy against virus infections. In hepatitis C virus (HCV), the sequence of the 5'UTR is conserved but the highly folded RNA structure severely limits the number of accessible sites. To expand investigation of targeting in the HCV genome, we have considered an internal genomic region whose sequence variation has been widely investigated and which has a particularly conserved RNA structure, which makes it accessible to the human RNase P in vitro. We have first mapped the accessibility of the genomic RNA to complementary DNAs within this internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P M1 RNA guide sequence ribozymes targeted to the selected sites. Considerations of RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack.

Base Sequence↗

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↗