Crispr Could Revolutionize the Way We Eradicate Invasive Species

How DNA Editing Could Change Life on Earth

Researchers hope to use the powerful biological tool to alter the genetic destiny of species and eliminate diseases.

AI-designed nucleases build on nature’s design

Researchers used AI to create variants of a CRISPR-Cas12-like nuclease, some of which show increased editing activity

(PDF) Highly efficient genome editing using oocyte-specific z cas9 transgenic zebrafish

PDF | Since its first application, CRISPR/Cas9 rapidly becomes a routine technique to perform genome editing in a variety of biological systems. To... | Find, read and cite all the research you need on ResearchGate

Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish. Liu Y, Zhang C, Zhang Y, Lin S, Shi DL, Shao M

Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish Since its first application to induce mutations in mammalian cells (Cong et al., 2013; Mali et al., 2013), CRISPR/Cas9 rapidly becomes a routine technique to perform genome editing in a variety of biological systems due to its facile, robust, and multiplexable features (Hwang et al., 2013; Guo et al., 2014; Wang et al., 2013). In every system, Cas9 and sgRNA should co-express to induce mutations. This is typically achieved by injection of the sgRNA mixed with Cas9 mRNA or protein into the fertilized eggs in zebrafish. As the in vivo translation of capped RNAs delay the presence of sufficient Cas9 protein, the mRNA injection produces more mosaic animals and shows relatively lower efficiency when compared to Cas9 protein injection (Burger et al., 2016). However, it is much more cost effective than using commercially available Cas9 protein, especially in the case of large-scale mutation screens. To further simplify this procedure, lower the cost and maintain an acceptable high genome editing efficiency in zebrafish, we set out to generate a transgenic line with stable and specific Cas9 expression in the egg. zpc (zp3b) promoter (zpc0.5) was reported to drive robust and specific GFP expression in the oocyte (Onichtchouk et al., 2003), we thus fused it with a zebrafish codon optimized cas9 (zcas9) followed by an SV40 3' UTR (Fig. 1A and File S1; Liu et al., 2014), and introduced this construct to zebrafish genome by Tol2 mediated transgenesis (Urasaki et al., 2006). To screen female F0 fish harboring this transgene, the F0 female founders were outcrossed with the wild-type male, the resulting eggs were injected with an sgRNA targeting slc45a2 (Moreno-Mateos et al., 2015), a gene essential for melanin synthesis, and assayed for their pigmentation phenotype. We found that the offspring of one founder showed a high proportion of pigmentation defect. Some of them exhibited a homozygous mutant like albino phenotype, suggesting a robust Cas9 expression in the egg and a very high efficient gene disruption. To examine the specificity of zcas9 expression in the transgenic line, the offspring (F1) of this F0 founder were raised to adulthood, and the ovaries of female F1 transgenic fish were dissected and subjected to in situ hybridization (ISH) employing the probe of zcas9. We found that the transcripts of zcas9 were specifically distributed in oocytes with a diameter of 40-200 µm, at around IB-II stages, while in WT embryos, no hybridization signals were detected (Fig. 1B, C). We did not detect zcas9 expression in stage IA oocytes or in larger ones after stage II. Accordingly, by RT-PCR, we also failed

Prime editing-installed suppressor tRNAs for disease-agnostic genome editing

Precise genome-editing technologies such as base editing1,2 and prime editing3 can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration4,5 or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.

ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish

Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish

Cas9 RNP complexes consisting of synthetic crRNA:tracrRNA duplex guide RNAs consistently induce mutations in virtually all copies of a targeted gene in zebrafish embryos. Hoshijima et al. show these tools allow effective screening of individual or combinations of gene function in F0 embryos and the facile induction of deletion mutations.

BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish

The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.

Stage- and tissue-specific gene editing using 4-OHT–inducible Cas9 in whole organism

Li et al. report a novel Cas9-based spatiotemporal gene-editing approach in zebrafish. Given the limited germline transmission efficiency of knock-in allel

The one science reform we can all agree on, but we're too cowardly to do

OR: the long overdue forest fire

Cytosine base editors with increased PAM and deaminase motif flexibility for gene editing in zebrafish

Cytosine base editing is a powerful tool for making precise single nucleotide changes in cells and model organisms like zebrafish, which are valuable for studying human diseases. However, current base editors struggle to edit cytosines in certain DNA contexts, particularly those with GC and CC pairs, limiting their use in modelling disease-related mutations. Here we show the development of zevoCDA1, an optimized cytosine base editor for zebrafish that improves editing efficiency across various DNA contexts and reduces restrictions imposed by the protospacer adjacent motif. We also create zevoCDA1-198, a more precise editor with a narrower editing window of five nucleotides, minimizing off-target effects. Using these advanced tools, we successfully generate zebrafish models of diseases that were previously challenging to create due to sequence limitations. This work enhances the ability to introduce human pathogenic mutations in zebrafish, broadening the scope for genomic research with improved precision and efficiency.

Egon Willighagen (@egonw@social.edu.nl)

I commented on @PubPeer@mastodon.social on "Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems" https://doi.org/10.3389/fbioe.2025.1669104, that got published today. I don't do this often, and even hesitant to post here. But I have some questions: https://www.pubpeer.com/publications/E18F253C8EB7B947220AFDB4F608FC#1 I welcome if other people can also have a look. I did some journal editing yesterday, and that makes me sometimes more critical than I need to be.

Strengthening biosecurity in the era of AI

AI is reshaping biology, unlocking breakthroughs while raising new risks. Learn how smarter safeguards can strengthen biosecurity without slowing innovation.

Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications

Agriculture is under pressure to provide food for a growing population and the feedstock required to drive the bioeconomy. Methods to breed and genetically modify plants are inadequate to keep pace. When engineering crops, traits are painstakingly introduced into plants one-at-a-time, combine unpredictably, and are continuously expressed. Synthetic biology is changing these paradigms with new genome construction tools, computer aided design (CAD), and artificial intelligence (AI). “Smart plants” contain circuits that respond to environmental change, alter morphology, or respond to threats. Further, the plant and associated microbes (fungi, bacteria, archaea) are now being viewed by genetic engineers as a holistic system. Historically, plant health has been enhanced by many natural and laboratory-evolved soil microbes marketed to enhance growth or provide nutrients, or pest/stress resistance. Synthetic biology has expanded the number of species that can be engineered, increased the complexity of engineered functions, controlled environmental release, and can assemble stable consortia. New CAD tools will manage genetic engineering projects spanning multiple plant genomes (nucleus, chloroplast, mitochondrion) and the thousands of genomes of associated bacteria/fungi. This review covers advanced genetic engineering techniques to drive the next agricultural revolution, as well as push plant engineering into new realms for manufacturing, infrastructure, sensing, and remediation.

Kindling — Regenerate Your Feed

Erase cruelty. Preserve critique.