Institute of Molecular and Cell Biology (IMCB)’s Post

How to measure the efficiency of RNA-guided genome editing in plants 🌱?   A quantitative assay using a Csy4-targeted system, where the Csy4 gene is deleted by CRISPR/Cas-induced double-strand breaks (DSBs). The assay's design allows the indirect quantification of full-gene deletions by measuring the reactivation of reporter genes, such as luciferase or fluorescent proteins, which are suppressed when Csy4 is present. Developed and published by scientists from LMU Munich and Leibniz Institute of Plant Biochemistry! Great work by my former co-worker Tom Schreiber ✨!   Components of the assay include: - A reporter gene suppressed by Csy4 cleavage ✂️. - Cas9 or Cas12a endonucleases that introduce DSBs 🧬 to delete the Csy4 gene. - Normalization using reference genes like Renilla luciferase or hygromycin phosphotransferase.   The assay 📊 was validated using Nicotiana benthamiana leaves 🌿 and Lotus japonicus calli 🧫, achieving over 90% correlation between reporter gene expression and successful Csy4 deletions. It demonstrated that Cas9 had higher editing efficiency than Cas12a, particularly in transient expression systems, while the difference diminished with stable transformations over time.   The assay can be adapted for various genome editing applications in different organisms and serves as a versatile platform for optimizing CRISPR/Cas systems. It offers significant advantages for comparing editing efficiencies and refining gene knockout strategies.   #CRISPR #GenomeEditing #PlantBiotechnology #Cas9 #Cas12a #RNAi #GeneEditing #AgriculturalResearch #MolecularBiology #BiotechInnovation #PlantScience #GeneticEngineering #CasSystems #ResearchInnovation #ScientificAdvances

Demeetra is excited to share our collaborative research effort with Indiana University which has led to a significant breakthrough in biopesticide development and is being recognized as an Editor’s Choice Article by the MDPI Journal. This innovative study harnesses the power of Cas-CLOVER and piggyBac gene editing technologies in yeast to forge a path towards safer, more effective biopesticides. The focus of Demeetra’s contributions to this work is the engineering of a yeast strain capable of enhanced stable RNA interference (RNAi) expression through scale-up fermentation, aimed at advancing the sterile insect technique (SIT) for mosquito control. Demeetra scientists led by Dr. Corey Brizzee, PhD, Director of Gene Editing, first used Cas-CLOVER targeted nuclease, a more precise alternative to CRISPR/Cas9, to knock out multiple genes that could be used to select for high expressing clones. Using the edited yeast strain as a foundation, piggyBac transposase was employed to stably deliver a wide range of gene copies at different integration sites. Demeetra's expertise in gene editing shines through this project, offering an accessible and precise alternative to conventional CRISPR/Cas9 methods. Our proprietary technologies, Cas-CLOVER and piggyBac, have demonstrated exceptional potential in broader applications across synthetic biology, bioprocessing and agriculture. Demeetra CEO, Jack Crawford, and Research Associate, Seth McConnell, were also recognized as authors on the paper, highlighting our commitment to exceptional transfer of gene editing know-how to our licensees through our peer-review published staff. Join us in exploring the vast potential of these technologies. See the full summary here: https://lnkd.in/eFNRJpW9 #GeneEditing #CRISPR #SyntheticBiology #AgriculturalBiotechnology #Bioeconomy

A University of Sydney team led by Sandro Ataide have developed a superior gene editing tool set to the current industry standard CRISPR. "SeekRNA uses a programmable ribonucleic acid (RNA) strand that can directly identify sites for insertion in genetic sequences, simplifying the editing process and reducing errors." https://lnkd.in/g4_5wakt #biology #genetics #geneticengineering #biotech #biotechnology #molecularbiology #cellbiology #biochemistry #computationalbiology

𝐇𝐨𝐰 𝐃𝐍𝐀 𝐂𝐨𝐩𝐲 𝐢𝐭𝐬𝐞𝐥𝐟? DNA replication is a vital biological process through which a cell makes an identical copy of its DNA, ensuring that genetic information is transmitted accurately during cell division. The fundamental mechanism of DNA replication is the unwinding of the double helix, followed by the synthesis of two new complementary strands through base pairing. This occurs via a series of well-coordinated steps, primarily involving enzymes such as #DNA helicase, DNA polymerase, and DNA ligase. 𝐂𝐥𝐢𝐜𝐤 𝐇𝐞𝐫𝐞 𝐭𝐨 𝐆𝐞𝐭 𝐅𝐫𝐞𝐞 𝐏𝐃𝐅 𝐁𝐫𝐨𝐜𝐡𝐮𝐫𝐞:  https://lnkd.in/eAvKm5jf #Plasmid DNA, a form of circular, double-stranded DNA found in many bacteria, plays a significant role in bacterial #genetics and biotechnology. Unlike chromosomal DNA, which is housed within the nucleus of eukaryotic cells, plasmids exist independently in the cytoplasm and can replicate autonomously. Plasmids often carry genes that confer advantageous traits, such as #antibiotic resistance, which can be spread among bacteria through horizontal gene transfer. The replication of plasmid DNA occurs through a process known as rolling circle replication. During this process, a single-stranded break in the circular plasmid initiates the replication. The free 3’ end of the broken strand serves as a primer for DNA polymerase, synthesizing new DNA and displacing the older strand, which is also replicated. This creates multiple copies of plasmid DNA. Additionally, plasmids are widely utilized in #molecular biology for cloning and genetic engineering. Scientists can insert specific genes into plasmids, introduce them into host bacteria, and utilize the host’s replication machinery to produce multiple copies of the gene or even express the gene product. This technique has been pivotal in producing recombinant proteins and developing gene therapies. Understanding the mechanisms of DNA replication, including that of plasmids, is crucial for advancements in genetic research, biotechnology, and medicine. #DNA #Plasmiddna #gene #therapy #therapeutics #medicine #medical #pharma #future #innovation 

𝐇𝐨𝐰 𝐃𝐍𝐀 𝐂𝐨𝐩𝐲 𝐢𝐭𝐬𝐞𝐥𝐟? DNA replication is a vital biological process through which a cell makes an identical copy of its DNA, ensuring that genetic information is transmitted accurately during cell division. The fundamental mechanism of DNA replication is the unwinding of the double helix, followed by the synthesis of two new complementary strands through base pairing. This occurs via a series of well-coordinated steps, primarily involving enzymes such as #DNA helicase, DNA polymerase, and DNA ligase. 𝐂𝐥𝐢𝐜𝐤 𝐇𝐞𝐫𝐞 𝐭𝐨 𝐆𝐞𝐭 𝐅𝐫𝐞𝐞 𝐏𝐃𝐅 𝐁𝐫𝐨𝐜𝐡𝐮𝐫𝐞: https://lnkd.in/eAvKm5jf #Plasmid DNA, a form of circular, double-stranded DNA found in many bacteria, plays a significant role in bacterial #genetics and biotechnology. Unlike chromosomal DNA, which is housed within the nucleus of eukaryotic cells, plasmids exist independently in the cytoplasm and can replicate autonomously. Plasmids often carry genes that confer advantageous traits, such as #antibiotic resistance, which can be spread among bacteria through horizontal gene transfer. The replication of plasmid DNA occurs through a process known as rolling circle replication. During this process, a single-stranded break in the circular plasmid initiates the replication. The free 3’ end of the broken strand serves as a primer for DNA polymerase, synthesizing new DNA and displacing the older strand, which is also replicated. This creates multiple copies of plasmid DNA. Additionally, plasmids are widely utilized in #molecular biology for cloning and genetic engineering. Scientists can insert specific genes into plasmids, introduce them into host bacteria, and utilize the host’s replication machinery to produce multiple copies of the gene or even express the gene product. This technique has been pivotal in producing recombinant proteins and developing gene therapies. Understanding the mechanisms of DNA replication, including that of plasmids, is crucial for advancements in genetic research, biotechnology, and medicine. #DNA #Plasmiddna #gene #therapy #therapeutics #medicine #medical #pharma #future #innovation

DNA replication #biology

The field of synthetic biology is at an inflection point – ready to accelerate the creation of personalized and cost-effective genetic medicines. Our CTO Phil Paik explains that at the foundation of this parardigm shift is the ability of new enzymatic DNA synthesis approaches to produce longer chains of DNA with higher accuracy than currently used chemical-based synthesis. Read the full article here: https://lnkd.in/daa84eQA #DNAuncompromised #enzymaticDNAsynthesis #DNAsynthesis #FES #DNA #CRISPR #geneediting #geneticmedicines #genomics #genetics #synbio #SynBioBeta2024

🔬 Did you know that selecting the appropriate competent strain can help maintain and purify the integrity of plasmids with extended poly(A) tails? 🌟 Explore how genetically engineered strains are revolutionizing molecular biology with cutting-edge technologies and innovative research. ✔️Discover advanced gene editing and high-throughput screening driving the creation of high-quality engineered strains. ✔️Essential for Research: Understand why researchers need to engineer strains for improved DNA cloning, plasmid construction, and protein expression in molecular biology. ✔️Learn how GenScript’s Poly(A) Strain V2/V3 enhances stability and efficiency, ensuring high-quality results in genetic research. 👉Read Now: https://ow.ly/6f3W50Savfy #cloning #strain #plasmid #molecularbiology #RelibailityisinourGENES

Happy to share the lab's new publication "Linking CRISPR–Cas9 double-strand break profiles to gene editing precision with BreakTag", appeared in @NatureBiotech couple of days ago. We describe BreakTag & BreakInspectoR, an NGS-based methodology along with the accompanied bioinformatics pipeline for the rapid, efficient & genome wide characterization of CRISPR nucleases activity and fidelity https://lnkd.in/dkKYWWkP

Evaluation of NGS DNA barcoding for biosecurity diagnostic applications: case study from banana freckle incursion in Australia Molecular diagnostics in combination with morphological identification is the method of choice for several cryptic microbial plant pathogens. For some diagnostic applications, traditional sequencing techniques can be time consuming, making them ill-suited for biosecurity incursion responses, where accurate results are needed in real time. More rapid next generation sequencing tools must be tested and compared with traditional methods to assess their utility in biosecurity applications. Here utilizing 95 samples infected with fungal pathogen Phyllosticta cavendishii, from a recent incursion in Australia, we compare species identification success using Internal Transcribed Spacer (ITS) gene barcode on conventional Sanger and Oxford Nanopore MinION sequencing platforms. For Sanger sequencing, the average pairwise identity percentage score between generated consensus sequences and P. cavendishii sequence from holotype material on NCBI database was 99.9% ± SE 0.0 whereas for MinION sequencing the average pairwise identity percentage was 99.1% ± SE 0.1. Relatively larger consensus sequences (mean 486 bp ± SE 2.4) were generated by Sanger sequencing compared to MinION sequencing (mean 435 bp ± SE 4.6). Our results confirm that both sequencing methods can reliably identify P. cavendishii. MinION sequencing, provided quicker results compared to Sanger sequencing and demonstrated diagnostic competence, with the added advantage of being portable, for front-line “point of incursion” biosecurity applications. https://lnkd.in/gPU_B27c