Novel Biotechnology Inc.’s Post

Does the integrity of your plasmid DNA keep you up at night? If not, maybe it should. A recent Nature article highlights results from a large-scale study on plasmid integrity, and the results are not good. The study examined 1000s of plasmids from labs around the world and uncovered a shocking result – most plasmids have design flaws or undetected sequence errors that are likely contributing to unexpected experimental failures. The study notes several common issues with plasmids: First, poor plasmid design on the part of the experimenter compromises plasmid functionality (like failing to add a stop codon to a gene). Second, the physical sequence of the plasmid does not match the corresponding electronic sequence map. These errors are due the outdated technology that many labs use to produce their plasmids coupled with poor quality control.  With a subset of plasmids subjected to sequencing in the study, the authors found that only 65% of provided plasmids matched their electronic plasmid map (meaning that 35% of plasmids either contained undesired mutations or were actually completely different plasmids than expected). Shockingly, when they examined plasmids used for gene therapy applications, they found that 36% of plasmids had mutations in one of the two ITR regions (sequences that play a critical role in the functionality of viral-based gene therapies), meaning that these plasmids will likely not function as intended. The authors concluded that “In total, we estimate that 45-50% of lab-made plasmids have undetected design and/or sequence errors that could potentially compromise the intended applications.” They further conclude that “The high error rate of lab-made plasmids suggests that many labs lack the sophisticated and nuanced expertise needed to properly design vectors and furthermore, there is insufficient quality control of the plasmids being constructed and propagated in labs.” Serving as CTO at a company developing new tech for plasmid manufacturing, I can tell you these results are not surprising, nor are they only a problem in academic labs. At Novel Biotechnology Inc., we perform extensive quality control on all the plasmids we work with, including plasmids that we receive from clients and outside vendors where undetected mutations are extremely common. In one extreme case, we analyzed a plasmid manufactured by another vendor for one of our clients and found that it was highly contaminated with multiple other plasmids which our client had never worked with – meaning the vendor had sent them a plasmid prep that was a mix of other customers’ plasmids! This not only compromised the experiments our client had planned, but given the highly proprietary nature of plasmid designs, could seriously compromise the IP position of those other companies! This wasn’t a small, unknown vendor either. This is a serious conversation that needs to be had, especially as genetic medicines rise in prominence.

Nature Magazine’s article “Serious errors plague DNA tool that’s a workhorse of biology” reveals significant flaws in plasmids, which are essential tools in biotherapeutic manufacturing and research. A study found that nearly half of the analyzed plasmids contained critical errors, potentially compromising experimental results. These errors often occur in sequences vital for gene expression and replication such as ITR regions and homopolymers. https://lnkd.in/dkpa9Avt   Unlike plasmid DNA, our enzymatically produced LineaDNA and can rapidly produce DNA constructs containing challenging sequences such as ITRs and homopolymers that have high failure rates in plasmids. These sequences are critical for the manufacture of important therapies such as gene and mRNA therapies. Learn more about our LinearDNA platform: https://linearxdna.com/

CRISPR- Cas9 has a scope to involve two g-RNAs instead of single g-RNA which can result into increased specificity.

𝐈𝐝𝐞𝐧𝐭𝐢𝐟𝐢𝐜𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐓𝐚𝐫𝐠𝐞𝐭 𝐃𝐍𝐀 𝐒𝐞𝐪𝐮𝐞𝐧𝐜𝐞𝐬 𝐛𝐲 𝐋𝐚𝐜 𝐑𝐞𝐩𝐫𝐞𝐬𝐬𝐨𝐫 & 𝐂𝐑𝐈𝐒𝐏𝐑-𝐂𝐚𝐬𝟗 𝐂𝐨𝐦𝐩𝐥𝐞𝐱   In molecular biology, the precise identification and manipulation of target DNA sequences are pivotal for various applications, including genetic engineering and gene therapy. Among the innovative methods, the combined use of the Lac repressor (LacI) and CRISPR-Cas9 complex emerges as a powerful tool for DNA sequence recognition and editing. The Lac repressor, derived from the lac operon of Escherichia coli, is renowned for its ability to specifically bind to the lac operator sequence. This binding is highly specific and forms the basis of the lac repressor's role in regulating gene expression. Leveraging this specificity, researchers have engineered synthetic systems where LacI serves as a DNA-binding domain, guiding the CRISPR-Cas9 complex to target #dna sequences with high precision. 𝐂𝐥𝐢𝐜𝐤 𝐇𝐞𝐫𝐞 𝐭𝐨 𝐆𝐞𝐭 𝐅𝐫𝐞𝐞 𝐏𝐃𝐅 𝐁𝐫𝐨𝐜𝐡𝐮𝐫𝐞: https://lnkd.in/eFWUHVy9 CRISPR-Cas9, an adaptive immune system found in bacteria and archaea, has been repurposed for gene editing in various organisms. The Cas9 nuclease, guided by a single-guide RNA (sgRNA), can be directed to specific DNA sequences for cleavage. By fusing the DNA-binding capability of LacI with the targeted DNA cleavage function of CRISPR-Cas9, researchers have created a potent tool for identifying and editing specific genomic loci. The fusion of LacI with #crispr -Cas9 allows for programmable and highly specific targeting of DNA sequences. Through the design of sgRNAs complementary to the target DNA flanked by Lac operator sequences, the Lac repressor recruits the CRISPR-Cas9 complex to the desired genomic sites. This dual recognition system enhances the accuracy and efficiency of DNA editing processes. The application of Lac repressor-guided CRISPR-Cas9 complexes extends across various fields, including gene knockout, gene activation, and base editing. Moreover, the modular nature of CRISPR-Cas systems enables further customization and optimization for specific experimental or therapeutic needs. In conclusion, the identification of target DNA sequences by the Lac repressor and CRISPR-Cas9 complex represents a sophisticated approach in #molecularbiology, enabling precise genome editing with broad implications for research and therapeutic interventions. The synergy between natural and engineered molecular components continues to drive advancements in #genetic manipulation techniques. #dnasequencing #crispr #crisprcas9 #dna #genetics

Plasmids are essential tools in biology labs for storing and exchanging genes, with the model organism Escherichia coli being a prime example. However, many lab-made #plasmids have problems. Researchers at VectorBuilder conducted a systematic evaluation of circular #DNA structures by analyzing more than 2,500 lab-produced plasmids sent to a company that packages them into viral vectors for gene therapy. Nearly half of the plasmids had design flaws, including errors in sequences critical to the expression of a therapeutic gene. Some plasmids contained genes encoding proteins toxic to E. coli, which could slow or stop the growth of the organisms biologists rely on to replicate their plasmids. Others encoded proteins toxic to these viruses, and some contained repetitive DNA sequences that can accumulate mutations within plasmids. Gene therapies are often packaged in adeno-associated viruses (AAVs), which are mostly harmless and can deliver genetic treatments to cells. About 40% of the #AAV plasmids in the study had mutations in the ITR regions that could corrupt this important message. If these faulty plasmids were used, the gene therapy might not work - and it could take scientists a long time to figure out why. Sequencing plasmids for errors could alert researchers to these problems, and some companies offer plasmid sequencing for about $15.00 per sample. It is also recommended that new lab members learn from experienced plasmid constructors to avoid wasting time and money.

#NatureBites - Laboratory-made plasmids often contain significant design flaws, as revealed by a systematic assessment of over 2,500 plasmids. Nearly half of these plasmids had errors in critical sequences, highlighting a widespread lack of proper quality control in plasmid design, which can lead to irreproducible scientific results. Some plasmids contained genes toxic to the expression system, or had repetitive sequences prone to mutations. About 40% of plasmids intended for AAV packaging had mutations in ITR regions, compromising the efficacy of gene therapies. https://lnkd.in/egdHTrr3

When all you have is a hammer, every problem is a nail. But what if the handle of your hammer is only half as long as you thought, or the head is made of Swiss cheese? It'll be harder to build anything, that's for sure. Plasmids are like hammers for #molecularbiology, general purpose tools that can be applied to all sorts of construction problems––from exploratory #biology to cell and gene therapy. I spent most of my doctoral training introducing deliberate mutations into plasmids, to better understand how single amino acid changes in my favorite transcription factor affected #brain development. Typically, this entailed accurately changing 1 or 2 nucleotides in a 8000-10000 base pair plasmid. My doctoral advisor insisted on very rigorous #QC for point mutants: Introduce the mutation by PCR; verify that your intended mutation is present by Sanger sequencing; finally, subclone the smallest possible unique restriction fragment from your mutant plasmid back into the parent vector, to ensure no surprise mutations were introduced along with the intended one. It was onerous, basically doubling the time it took to make each new plasmid because of the extra cloning. But after getting wonky results using plasmids from other labs that had "bonus" mutations, or tagged inserts in the wrong reading frame, or missing start/stop codons, I learned to appreciate this approach. We would sequence any plasmid received from another lab, and often clone the inserts into our own expression vectors. Before Addgene existed for plasmid archiving and distribution, or services like Integrated DNA Technologies or Twist Bioscience offered low-cost gene synthesis, it was tacitly acknowledged that getting a plasmid from another lab was like opening Forrest Gump's proverbial box of chocolates. You never knew what you were going to get. In a BioRxiv preprint highlighted by Nature Portfolio this week, a team led by Bruce T. Lahn from VectorBuilder says the quiet part out loud, and does the field a huge service. They found roughly 1 in 7 lab-generated plasmids have structural differences from the nominal design, and as many as 1 in 3 have sequence mismatches. Worse still, they think this is an UNDERestimate of the true rate of inaccurate plasmid reporting. And while funky hammers might get the job done, these kind of errors introduce unanticipated variability into every new experiment. Of course, the grad students and postdocs making plasmids in academic labs are underpaid, overworked, and pressured to publish provocative results in fancy journals, so maybe it shouldn't come as a surprise that many plasmids are chock full o' errors? But applying some simple and dull QC practices to our basic tools would go a long way in addressing our ongoing scientific #reproducibility crisis. https://lnkd.in/gCD3Aj-Y https://lnkd.in/ggK7YTTt

A decade ago, CRISPR gene editing burst onto the global stage through a groundbreaking paper in Science. Eight years later, its senior authors, Jennifer A. Doudna and Emmanuelle Charpentier, were awarded the Nobel Prize in Chemistry. At that point, the transformative power of the CRISPR-Cas9 gene editing tool had already become evident, revolutionizing researchers' capacity to make precise modifications to genomes. CRISPR has proven its versatility in manipulating the genetic codes of various organisms, including humans and animals, thus reshaping the landscape of genetic research In addition to making it simpler to create more accurate model systems, gene editing with CRISPR has also changed the game when it comes to time. Before, making complex changes to genes for studying diseases took a long time (months or even years). But with CRISPR, these changes can happen in just weeks. Nowaday, we can create transgenic mice quickly and with high precision. These mice help us test and predict how well a drug might work and how safe it is, right from the beginning of the research. This speeds up the whole process and makes it more efficient. The future of animal models is intricately tied to the evolving landscape of CRISPR technology. CRISPR's unparalleled ease and precision in genome editing offer unprecedented control, influencing various stages of the drug development process. Despite limitations, such as off-target effects and challenges in creating intricate genome modifications, CRISPR-Cas9 holds great promise for advancing innovative medical applications. This technology could potentially address and even cure conditions ranging from cardiovascular disease to rare inherited blood disorders. Animal models remain crucial for driving research and discovery in these therapeutic developments. However, by ensuring that in vitro models possess the most relevant genetic and cellular characteristics before transitioning to animal testing, CRISPR-Cas9 genome engineering enhances the translational capacity of in vivo model systems. This not only boosts success rates but also contributes to reducing the overall reliance on animal experimentation #Animal_disease_model #Biomedical_Research #Drug_discovery_and_development #Understanding_Disease_With_CRISPR_Animal_Models poor mice and rats :/ Photo by Kai Pfaffenbach / Reuter's

New mechanism for regulating cell division in the bacterial pathogen Klebsiella uncovered. Under the microscope, the Klebsiella pneumoniae bacterium typically appears in the form of short rods (left). However, if the DNA of the bacteria is damaged, the cells continue to grow in the course of the stress response and long bacteria develop (r.). Klebsiella pneumoniae is one of the most common and most dangerous bacterial pathogens impacting humans, causing infections of the gastrointestinal tract, pneumonia, wound infections, and even blood poisoning. With the aim of discovering therapeutically exploitable weaknesses in Klebsiella,has taken a close look at the molecular biology of the bacteria and was able to uncover the importance of a small, non-coding ribonucleic acid (sRNA for short) for the gene regulation of K. pneumoniae. "Klebsiella is a relevant bacterium for research for several reasons. On the one hand, this bacterium is problematic in the clinic because Klebsiella is very adaptable, able to multiply rapidly, and continuously acquire further resistances in addition to the existing natural resistances to various antibiotic agents." "On the other hand, little is known about gene regulation in Klebsiella, especially in comparison to closely related species such as E. coli or Salmonella,".Together with a team of scientists from the Cluster of Excellence, she analyzed the transcriptome of Klebsiella in search of previously unknown sRNAs and clues to their functions. "In addition to many sRNAs that were already known from related bacteria, we also found over 50 new potential regulators,"."Balance of the Microverse" in Jena. The researchers determined the interaction partners of all these sRNAs using a method based on high-throughput sequencing. Autonomous cell division control When analyzing the RNA pairings identified in this way, the sRNA DinR aroused the particular interest of the researchers. "We were finally able to find out that DinR is produced by the cell when DNA damage occurs. Under this condition, DinR inhibits the formation of FtsZ, a structural protein that is important for cell division," says Ruhland. DinR thus controls a further, previously unknown mechanism with which the bacteria can interrupt cell division if there are defects in the genetic material. This serves to give the cell time to repair the damaged genome before it is passed on to another generation of the bacterium—from an evolutionary perspective, a mechanism that serves to produce offspring that are as healthy as possible. #pathogen #Klebsiella #dna #bacteria #protein #cell #genome #sRNA

RNA genes have many functions but a large proportion entail gene regulation-related functions that fall within the category of epigenetics. Mattick identifies multiple types of RNAs that perform these important functions: Small regulatory RNAs (e.g., microRNAs) which regulate translation of proteins, regulate epigenetic process, and are also involved in alternative splicing. Long non-coding RNAs (called “lncRNAs”) which also influence gene expression by controlling transcription factors and transcription-splicing and also modulate many genetically variable traits. Some may even encode peptides. Transposable elements are important for gene structure and function, and gene regulatory networks. Overall these types of functional RNAs undergo much post-transcriptional editing and are important for brain function, and also can foster “transgenerational epigenetic inheritance.” #junkdna #junkrna #players #evolution