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

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

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

Genetic circuits are a fundamental aspect of synthetic biology and play a crucial role in the development of microbial cell factories. These circuits are designed to control and optimize the metabolic pathways within microorganisms, enabling them to produce valuable chemicals, enzymes, and pharmaceuticals more efficiently. Metabolic Flux Optimization: Genetic circuits can be engineered to regulate metabolic flux, which is the rate at which substrates are converted into products in a metabolic pathway. By optimizing this flux, cell factories can increase the yield of desired products. Tolerance and Mutant Screening: Genetic circuits help in developing strains with higher tolerance to industrial and in screening mutants with enhanced production capabilities. Dynamic Regulation and High-Throughput Screening: The use of genetic circuits allows for the dynamic regulation of gene expression, which can balance cell growth and product synthesis. #Synthetic_biology #Genetic_circuits #Cell_factory https://lnkd.in/dX3eHuzf

DNA replication #biology

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

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