Genome Editing Tools

New from Arc Institute: Megabase scale genome-editing in human cells. Bridge recombinases are a new class of genome editors. In this paper, they were used to invert up to 920,000 base pairs of DNA and also cut out 130,000 base pairs, "with no apparent distance dependency." Let's look at what these Bridge Recombinases are, and why I think this is a big deal. There are dozens of CRISPR-based gene-editing tools that can be used to swap nucleotides in the genome, delete parts of genes, or replace sequences entirely. But they all rely on cell repair pathways to make their edits. If a researcher wants to permanently “shut down” a gene (using CRISPR-Cas9, for example), then Cas9 must go into the genome and make a cut at the position indicated by its guide RNA. The Cas9 doesn't then fix the DNA it has broken, however; the cell must do that a different way. There are two main ways to fix this damage. Non-homologous end joining quickly slaps the two broken strands together, often adding or deleting random bits of DNA in the process. For the second option, homology-directed repair, scientists can introduce a DNA "donor template" into cells alongside CRISPR-Cas9, and then the cell copies from this template to fix the break. But homology-directed repair only works reliably during specific phases of the cell cycle and happens less frequently than non-homologous end joining. Because CRISPR-based tools rely on these cellular repair pathways, edits are inherently unpredictable. Different cells will fix the damage in different ways, leading to different edits. (Prime editors get around these two major repair pathways, but rely on a third mechanism, called the mismatch repair pathway.) Here's the punchline: Bridge recombinases can make megabase-scale edits in human cells, and do so **without relying on cellular repair mechanisms,** which could make them more predictable and versatile in the long run. This Bridge genome-editing tool is also made from two parts: a protein (called the recombinase) that cuts and rejoins strands of DNA, and a RNA molecule (called the ‘Bridge’) that guides the recombinase to a specific location in a cell’s genome. One immediate application for these Bridge recombinases, given their ability to make megabase-scale rearrangements, is to make cancer cell lines. Many cancers are caused by large-scale genome rearrangements that can't easily be replicated with other genome-editing tools. Chronic myeloid leukemia, for example, happens when chunks of chromosomes 9 and 22 swap places. Using Bridge recombinases, researchers could recreate this rearrangement in healthy cells to study how it causes disease and how to reverse it. Ditto for Ewing’s sarcoma, a bone cancer caused by another type of chromosome fusion. TL;DR: This seems, to me, to be the first tool good enough to reliably rewrite large stretches of the human genome.

🌟AVAILABLE NOW🌟: A toolkit optimized for CRISPR-based genome editing in barley and wheat using both Cas9 and Cas12a systems. Thanks to a Research Team from the John Innes Centre. The toolkit maximizes transformation and editing efficiencies, enabling highly effective mutagenesis. It is designed to be accessible to the broader cereal research community (plasmids available on Addgene) and supports both simplex and multiplex genome editing applications.   Lawrenson, T., Clarke, M., Kirby, R. et al. An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat. Plant Methods 20, 123 (2024). https://lnkd.in/eUVs7yeX   Summary: The CRISPR Cas9 and Cas12a systems are widely used for plant genome editing 🌱🧬✂️. The researchers optimized these systems for barley and wheat, two important monocot crops. Various components such as codon optimization, intron inclusion, and guide RNA expression architectures were tested to identify the most effective configurations for efficient mutagenesis.   Results 🎯: Cas9 Optimization: A Zea mays (corn) codon-optimized Cas9 variant with 13 introns performed best in barley, editing 100% of T0 plants at three target genes simultaneously. In wheat, over 90% of T0 plants were edited at all three subgenomic targets.   Cas12a Optimization: The best Cas12a results were achieved with an Arabidopsis codon-optimized sequence containing 8 introns and a tRNA-based multiguide array. This system resulted in up to 90% mutation efficiency across three target genes in barley and wheat.   Intron Contributions: The study highlighted that not all introns contribute equally to mutagenesis efficiency, but multiple introns together significantly boost Cas12a activity.   Multiplex Editing: The toolkit allows for multiplex editing, enabling researchers to target multiple genes simultaneously. The Cas12a system, in particular, showed a synergistic effect when combining the D156R mutation and introns, achieving higher efficiency than either feature alone.   ⚙️Toolkit 🛠️: A modular cloning system based on GoldenGate assembly was developed for Cas9 and Cas12a systems. This system is available through AddGene, offering proven nuclease and guide expression cassette options that facilitate highly efficient genome editing in barley and wheat.   #CRISPR #GenomeEditing #Cas9 #Cas12a #OpenAccess #Barley #Wheat #PlantBiotechnology #CerealCrops #MultiplexEditing #GeneticEngineering #GoldenGate #IntronMediatedEnhancement #PrecisionBreeding

Nature Biomedical Engineering paper (30 Apr 26) introduced PRIME-In, a DSB-free genome editing platform that achieves targeted integration of large DNA payloads into primary human T cells without triggering double-strand breaks. The name stands for Prime Editing-Mediated Large Integration, and the results make a compelling case that the non-viral CAR T manufacturing problem may finally have a solution that works at clinical scale. 🔅 The core innovation: Instead of relying on HDR or HMEJ donor templates that require a DSB to initiate repair, PRIME-In uses a prime editor to nick the genomic target strand while simultaneously priming a donor plasmid with a short microhomology sequence (~35 nt) via reverse transcription. That primed microhomology hybridizes to the open genomic nick and recruits endogenous DNA polymerases to extend directly from the donor — no break, no recombinase, no two-step landing pad insertion. 🔅 PRIME-In 2.0 adds a second genomic nick via an extra guide RNA, achieving up to 88% knock-in in HEK293T cells and payloads as large as 9.2 kb at over 80% efficiency. 👍 In primary human T cells, historically the hardest target for non-viral editing, the platform achieved approximately 50% integration efficiency for a 3-kb CD19 CAR construct with an 11-fold T cell expansion over input in just 7 days. The team solved DNA toxicity by co-delivering mRNAs encoding two HCMV-derived viral proteins (innate immune and apoptosis antagonists), UL36 and UL37x1, alongside A151 (a DNA sensor inhibitor), pushing T cell viability from ~34% to over 81% post-electroporation, the first use of viral immune evasion proteins as a transient manufacturing adjuvant in T cell engineering. 🔅 The safety data is equally compelling: chromosomal translocations at just 0.2–0.4% versus 2.34% for HMEJ, off-target knock-in events under 4% genome-wide versus 23% for HMEJ, and PRIME-In-engineered CAR T cells clearing Raji tumors in NXG xenograft mice comparably to lentiviral CAR T counterparts. 📢 For comparison, QuadPE (Nature, April 2026) demonstrated a four-pegRNA prime assembly approach capable of integrating up to 26 kb in non-dividing cells, though primary T cell manufacturing data at this scale has not yet been reported for that platform. 👍 Non-viral T cell therapy manufacturing has been waiting for a platform that is simultaneously safe, efficient, scalable, and recombinase-free. PRIME-In is the most complete candidate to that challenge published to date. 🔅 Link (not full text): https://lnkd.in/gmM5zBcb 🔅 Patent documents: not surfaced yet. 🔅 Related Patent documents by the authors: US20240307539A1, US20220008464A1, US20230144097A1, US20190093125A1, US20160186208A1 #GeneEditing #CARTcell #PrimeEditing #Biotech

This newsletter explores the application of next-generation CRISPR and base editing for precise gene correction in genetic diseases, focusing on the latest breakthroughs in prime editing, mitochondrial genome correction, AI-driven guide RNA optimization, in vivo CRISPR delivery, and epigenome editing. Prime Editing 3.0 improves the precision of correcting complex mutations, while CRISPR base editing for mitochondrial DNA brings new hope for previously untreatable mitochondrial diseases. AI-driven gRNA design minimizes off-target effects and improves the safety of genome editing. Meanwhile, engineered nanoparticles enable targeted CRISPR delivery, making gene therapy more efficient. In addition, CRISPR-based epigenetic editing allows reversible gene regulation, opening up new possibilities for precision medicine. With these cutting-edge innovations, CRISPR and base editing technologies are advancing gene therapies for genetic diseases, cancer, and neurological diseases, bringing us closer to curative treatments. Join us and stay informed about the future of gene editing and personalized medicine! #CRISPR #GeneEditing #BaseEditing #PrimeEditing #GeneticTherapy #PrecisionMedicine #SyntheticBiology #AIinBiotech #GenomeEngineering #BiotechBreakthroughs #CSTEAMBiotech

Indian scientists from the CSIR-Institute of Genomics and Integrative Biology (IGIB) in New Delhi, in collaboration with the LV Prasad Eye Institute, have developed an advanced CRISPR gene-editing system using the FnCas9 enzyme. This new system is more precise and efficient than existing CRISPR-Cas9 technologies, significantly reducing unintended DNA damage. CRISPR technology allows researchers to edit genes by targeting specific DNA sequences. However, current systems, like SpCas9, often face issues with off-target effects and reduced efficiency. The IGIB team has engineered new versions of FnCas9 that show higher accuracy and lower off-target effects. The researchers demonstrated the efficacy of their system on induced pluripotent stem cells from individuals with the RPE65 mutation, associated with inherited blindness. The enhanced FnCas9 successfully corrected the mutation in these cells, showing potential for therapeutic applications. This breakthrough is poised to have significant implications for gene therapy, especially in treating genetic disorders such as inherited blindness. The team aims to patent their innovation, emphasizing its potential to offer cost-effective solutions for gene editing in countries like India. #CRISPR

In vivo human T cell engineering with enveloped delivery vehicles Vertex Biopharm Consulting (https://lnkd.in/eu5EU_CS) Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility. https://lnkd.in/eqKFtpkk

Ever wonder how we got seedless watermelons, disease-resistant wheat, and tomatoes that stay fresh for weeks? It wasn't luck. It was precision science using multiple techniques, each solving a different agricultural challenge. For thousands of years, farmers selected the best plants and bred them together, hoping for improvement. Building the next generation of resilient crops relies on several distinct modification techniques. Here is a look at the actual science driving modern agriculture: 🔹 Cross-breeding combines two sexually compatible species to merge desirable parent traits. This traditional method gave us modern corn from ancient teosinte. 🔹 Polyploidy multiplies chromosome sets to impact fertility and size. This is why seedless grapes exist and why some strawberries are massive compared to their wild ancestors. 🔹 Mutagenesis uses controlled mutagens like targeted radiation to induce random mutations, then selects for beneficial traits. Sounds dramatic, but it's created hundreds of crop varieties we eat daily, including your Ruby Red grapefruit. 🔹 Protoplast Fusion merges cells or cell components from different species, transferring traits that couldn't naturally cross-breed. Think of it as cellular matchmaking beyond sexual compatibility barriers. 🔹 Transgenesis adds genes from completely different species to create varieties with desired traits. Inserting bacterial genes into corn made it pest-resistant (Bt corn), reducing pesticide needs dramatically. 🔹 Genome Editing uses enzyme systems like CRISPR to modify DNA directly within the cell with surgical precision. No foreign genes added, just targeted edits to the plant's own genome. It's how we're creating disease-resistant cacao to save chocolate from extinction. Each technique serves a purpose. Cross-breeding is slow but natural. Genome editing is fast and precise. Transgenesis crosses species barriers. Mutagenesis introduces controlled randomness. The question isn't whether we should modify crops—we've been doing it for 10,000 years. The question is: which tool for which challenge? As we look toward the future of climate-adapted agriculture, which of these technologies do you believe will drive the most critical commercial breakthroughs over the next decade? Drop your reaction below. 👇 #plantscience #cropmodification #agriculturalinnovation #crispr #plantbreeding #biotechnology #foodsecurity #genomeediting #agtech

🧬 #𝗟𝗡𝗣𝘀 𝗳𝗼𝗿 𝘁𝗵𝗲 𝗱𝗲𝗹𝗶𝘃𝗲𝗿𝘆 𝗼𝗳 𝗽𝗿𝗼𝘁𝗲𝗶𝗻-𝗥𝗡𝗔 𝗰𝗼𝗺𝗽𝗹𝗲𝘅𝗲𝘀 𝗳𝗼𝗿 𝗴𝗲𝗻𝗲 𝗲𝗱𝗶𝘁𝗶𝗻𝗴? ◽ 𝗖𝗵𝗲𝗻 𝗲𝘁 𝗮𝗹. 𝗶𝗻 𝗡𝗮𝘁𝘂𝗿𝗲 𝗕𝗶𝗼𝘁𝗲𝗰𝗵𝗻𝗼𝗹𝗼𝗴𝘆 (𝗟𝗶𝗻𝗸 𝗶𝗻 𝗰𝗼𝗺𝗺𝗲𝗻𝘁𝘀) led by Jennifer A. Doudna from University of California, Berkeley engineered a thermostable Cas9 (iGeoCas9), which was co-delivered as iGeoCas9 (protein!) RNP-LNP complex with sgRNA for genome editing in vivo (instead of delivering Cas9 encoding mRNA/DNA). ◽𝗖𝗼𝗻𝘁𝗲𝗻𝘁 Delivering CRISPR ribonucleoproteins (RNPs) using LNPs could enable efficient, low-toxicity, and scalable genome editing in vivo, provided that effective RNP-LNP complexes can be produced. The team engineered a thermostable Cas9 from Geobacillus stearothermophilus to create iGeoCas9 variants. These variants achieved over 100-fold more genome editing in cells and organs compared to native GeoCas9. iGeoCas9 RNP-LNP complexes could edit various cell types and promote homology-directed repair (HDR) in cells that received co-delivered ssDNA templates. Using tissue-specific LNP formulations, the team observed genome editing levels of 16-37% in the liver and lungs of reporter mice after a single IV injection of iGeoCas9 RNP-LNPs. iGeoCas9 RNPs combined with biodegradable LNPs edited the disease-causing SFTPC gene in lung tissue with an average efficiency of 19%, significantly improving upon previous genome editing levels achieved with viral or non-viral delivery methods. ◽ 𝗧𝗮𝗸𝗲𝗮𝘄𝗮𝘆 One of the rare examples in which LNPs are able to efficiently deliver protein-RNA complexes (hope to see more of that in the future). ◽ 𝗚𝗲𝗻𝗲 𝗗𝗲𝗹𝗶𝘃𝗲𝗿𝘆 ｜ 𝗖𝗲𝗹𝗹 & 𝗚𝗲𝗻𝗲 𝗧𝗵𝗲𝗿𝗮𝗽𝘆 𝗡𝗲𝘄𝘀 Follow me for more breaking research & news.

Excited to share our new paper in Nature Biotechnology: "Optimized R2 retroelement complexes for DNA insertion into plant genomes." 📄 https://lnkd.in/eva2n-c6 Inserting genes into plant genomes usually relies on Agrobacterium, which integrates DNA at random, leading to variable expression, silencing, and generations of screening to find a usable line. Targeted alternatives like CRISPR-Cas9 HDR and prime editing help, but are limited by efficiency or how much DNA they can carry. We took a different route: we repurposed an R2 retrotransposon from the zebra finch into a plant genome editor. R2 is a retroelement that inserts DNA into the 25S ribosomal DNA using its own RNA as a template, via target-primed reverse transcription. The 25S rDNA is an ideal "safe harbor": present in many copies, highly transcribed, and free of essential genes, so inserted genes get steady, predictable, high expression. What we found: • ~1 integrated copy per genome on average, ~90% full-length, about 30× more efficient than Cas9 HDR at the same site • Large cargo: a >5-kb, 3-enzyme metabolic pathway, with no drop in efficiency from 2 to 5 kb (so likely room for more) • High specificity & precision: ~96% of insertions at the target rDNA, predominantly seamless junctions  • No silencing: inserted payloads stayed unmethylated, with persistent expression • Works across species: Arabidopsis, N. benthamiana, and tomato The system is designed to slot into existing transformation pipelines: in place of your Cas9/donor, deliver the R2 protein + payload plasmid for targeted, multi-kb insertion at the rDNA safe harbor. If you are interested in testing the system in your species or application, feel free to reach out. 📩 Congratulations to lead author Kimberley Muchenje and the whole team!

MD Anderson Cancer Center just used CRISPR to supercharge NK cell therapy. Their new PreCiSE platform is the first genome-wide CRISPR screen built specifically for natural killer cells—and the results are wild: → Deleting genes like MED12, ARIH2, and CCNC turned NK cells into cancer killers → CAR-NK function and metabolic fitness shot up → Even treatment-resistant models responded Why it matters: We’ve known CRISPR can rewrite biology. Now it’s rewriting immunotherapy. T cells have dominated the headlines, but NK cells are faster, safer, and don’t require patient-by-patient engineering. With PreCiSE, we finally have the blueprint to make them just as powerful. This isn’t just a tool—it’s a turning point. CRISPR isn’t only editing the genome. It’s editing the future of cancer therapy. Question is: who will run with this first—the big pharmas, or the hungry biotechs?