#CRISPR-based genome editing

[blueoaknx]

Mitochondrial Dysfunction in mtARS Disorders

Introduction

Mitochondria are indispensable organelles that facilitate cellular bioenergetics, predominantly through oxidative phosphorylation (OXPHOS). Mitochondrial aminoacyl-tRNA synthetases (mtARS) are essential for the fidelity of mitochondrial translation, catalyzing the ligation of amino acids to their cognate tRNAs. Mutations in mtARS genes precipitate a spectrum of mitochondrial disorders, culminating in dysfunctional protein synthesis and aberrant mitochondrial bioenergetics. This review delves into the molecular pathogenesis of mitochondrial dysfunction in mtARS disorders, elucidating their biochemical perturbations, clinical phenotypes, and emerging therapeutic paradigms.

Molecular Pathophysiology of mtARS Disorders

MtARS enzymes ensure translational accuracy by charging mitochondrial tRNAs with their respective amino acids, a prerequisite for mitochondrial protein biosynthesis. Pathogenic variants in mtARS genes result in defective aminoacylation, perturbing mitochondrial translation and compromising the integrity of the electron transport chain (ETC). These perturbations induce bioenergetic deficits, increased reactive oxygen species (ROS) production, and secondary mitochondrial stress responses, leading to cellular demise.

Genetic Etiology of mtARS Mutations

Dysfunctional mtARS genes such as DARS2, AARS2, RARS2, and YARS2 have been implicated in autosomal recessive mitochondrial disorders. These mutations exhibit tissue-specific phenotypic heterogeneity, with neurological, muscular, and systemic manifestations. For instance, DARS2 mutations drive leukoencephalopathy with brainstem and spinal cord involvement, whereas AARS2 defects result in a constellation of neurodegenerative and ovarian pathologies.

Biochemical and Cellular Consequences

Dysfunctional mtARS enzymes manifest in multifaceted mitochondrial deficits, including impaired translation, defective OXPHOS, and dysregulated mitochondrial proteostasis.

Disruption of Mitochondrial Translation

Impaired aminoacylation abrogates the synthesis of mitochondrially encoded proteins, undermining the assembly of ETC complexes. This translational arrest culminates in defective ATP synthesis and precipitates a systemic energy deficit.

Electron Transport Chain Dysfunction and Bioenergetic Failure

Pathogenic mtARS mutations lead to OXPHOS inefficiencies, reducing mitochondrial membrane potential (Δψm) and ATP output. Perturbed electron flux exacerbates ROS accumulation, instigating oxidative damage and apoptotic cascades.

Mitochondrial Unfolded Protein Response (UPRmt) Activation

Cellular compensatory mechanisms, including UPRmt, are upregulated in response to mitochondrial translation failure. UPRmt mitigates proteotoxic stress via chaperone-mediated protein refolding and degradation pathways. However, chronic UPRmt activation fosters maladaptive stress responses, contributing to progressive cellular degeneration.

Clinical Manifestations

mtARS disorders exhibit phenotypic variability, spanning from mild neuromuscular impairment to severe multisystemic involvement. The pathophysiological hallmark includes disrupted neurological, muscular, and cardiac function.

Neurological Dysfunction

Neurodegeneration is a predominant feature of mtARS disorders, manifesting as ataxia, seizures, intellectual disability, and progressive leukoencephalopathy. Magnetic resonance imaging (MRI) frequently reveals white matter abnormalities, indicative of compromised oligodendrocyte function.

Myopathy and Metabolic Dysregulation

Muscle tissue, with its high ATP demand, is particularly susceptible to mitochondrial dysfunction. Clinical hallmarks include hypotonia, muscle weakness, and exercise intolerance, often concomitant with metabolic anomalies such as lactic acidosis and elevated pyruvate-to-lactate ratios.

Cardiomyopathy and Mitochondrial Energetics

Hypertrophic cardiomyopathy has been observed in YARS2-associated mitochondrial disorders, wherein compromised ATP synthesis in cardiomyocytes disrupts contractile function and electrophysiological stability.

Diagnostic and Functional Evaluation

A combination of genomic, biochemical, and imaging modalities facilitates the diagnosis of mtARS disorders.

Genomic and Transcriptomic Analysis

Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are pivotal for identifying pathogenic mtARS variants. Transcriptomic profiling elucidates perturbations in mitochondrial gene expression networks, further refining diagnostic accuracy.

Functional Mitochondrial Assays

Biochemical assays, including high-resolution respirometry, ATP quantification, and ETC enzymatic profiling, provide insights into mitochondrial bioenergetics. Patient-derived fibroblasts and induced pluripotent stem cells (iPSCs) serve as valuable models for functional interrogation.

Neuroimaging and Biomarker Identification

Advanced imaging modalities such as MR spectroscopy (MRS) detect metabolic derangements, including lactate accumulation in affected brain regions. Circulating mitochondrial-derived peptides and metabolomic signatures are emerging as potential diagnostic biomarkers.

Emerging Therapeutic Strategies

Despite the absence of curative therapies, multiple avenues are under investigation to ameliorate mitochondrial dysfunction in mtARS disorders.

Mitochondria-Directed Antioxidants

Therapeutic compounds such as MitoQ, idebenone, and edaravone aim to attenuate oxidative stress and preserve mitochondrial integrity.

Genetic and RNA-Based Interventions

Gene therapy strategies utilizing adeno-associated virus (AAV)-mediated delivery and CRISPR-based genome editing are being explored for genetic correction of mtARS mutations. Additionally, RNA-based approaches, including antisense oligonucleotides (ASOs) and mRNA replacement therapy, hold promise in restoring mtARS functionality.

Metabolic Modulation and Supportive Therapies

Ketogenic diets, NAD+ precursors (e.g., nicotinamide riboside), and mitochondrial biogenesis activators (e.g., PGC-1α modulators) are under investigation to enhance cellular energy metabolism. Supportive interventions, including physical therapy and neuromuscular rehabilitation, remain integral to patient management.

Conclusion and Future Directions

Mitochondrial dysfunction in mtARS disorders arises from defective mitochondrial translation, OXPHOS perturbation, and maladaptive stress responses. Advances in genomic medicine, mitochondrial therapeutics, and precision medicine approaches are poised to transform the diagnostic and therapeutic landscape. Continued research into mtARS pathobiology, coupled with translational innovations, will be instrumental in developing targeted interventions for affected individuals.

[jcmarchi]

Killing the messenger

New Post has been published on https://thedigitalinsider.com/killing-the-messenger/

Killing the messenger

Like humans and other complex multicellular organisms, single-celled bacteria can fall ill and fight off viral infections. A bacterial virus is caused by a bacteriophage, or, more simply, phage, which is one of the most ubiquitous life forms on earth. Phages and bacteria are engaged in a constant battle, the virus attempting to circumvent the bacteria’s defenses, and the bacteria racing to find new ways to protect itself.

These anti-phage defense systems are carefully controlled, and prudently managed — dormant, but always poised to strike.

New open-access research recently published in Nature from the Laub Lab in the Department of Biology at MIT has characterized an anti-phage defense system in bacteria, CmdTAC. CmdTAC prevents viral infection by altering the single-stranded genetic code used to produce proteins, messenger RNA.

This defense system detects phage infection at a stage when the viral phage has already commandeered the host’s machinery for its own purposes. In the face of annihilation, the ill-fated bacterium activates a defense system that will halt translation, preventing the creation of new proteins and aborting the infection — but dooming itself in the process.

“When bacteria are in a group, they’re kind of like a multicellular organism that is not connected to one another. It’s an evolutionarily beneficial strategy for one cell to kill itself to save another identical cell,” says Christopher Vassallo, a postdoc and co-author of the study. “You could say it’s like self-sacrifice: One cell dies to protect the other cells.”

The enzyme responsible for altering the mRNA is called an ADP-ribosyltransferase. Researchers have characterized hundreds of these enzymes — although a few are known to target DNA or RNA, all but a handful target proteins. This is the first time these enzymes have been characterized targeting mRNA within cells.

Expanding understanding of anti-phage defense

Co-first author and graduate student Christopher Doering notes that it is only within the last decade or so that researchers have begun to appreciate the breadth of diversity and complexity of anti-phage defense systems. For example, CRISPR gene editing, a technique used in everything from medicine to agriculture, is rooted in research on the bacterial CRISPR-Cas9 anti-phage defense system.

CmdTAC is a subset of a widespread anti-phage defense mechanism called a toxin-antitoxin system. A TA system is just that: a toxin capable of killing or altering the cell’s processes rendered inert by an associated antitoxin.

Although these TA systems can be identified — if the toxin is expressed by itself, it kills or inhibits the growth of the cell; if the toxin and antitoxin are expressed together, the toxin is neutralized — characterizing the cascade of circumstances that activates these systems requires extensive effort. In recent years, however, many TA systems have been shown to serve as anti-phage defense.

Two general questions need to be answered to understand a viral defense system: How do bacteria detect an infection, and how do they respond?

Detecting infection

CmdTAC is a TA system with an additional element, and the three components generally exist in a stable complex: the toxic CmdT, the antitoxin CmdA, and an additional component called a chaperone, CmdC.

If the phage’s protective capsid protein is present, CmdC disassociates from CmdT and CmdA and interacts with the phage capsid protein instead. In the model outlined in the paper, the chaperone CmdC is, therefore, the sensor of the system, responsible for recognizing when an infection is occurring. Structural proteins, such as the capsid that protects the phage genome, are a common trigger because they’re abundant and essential to the phage.

The uncoupling of CmdC exposes the neutralizing antitoxin CmdA to be degraded, which releases the toxin CmdT to do its lethal work. 

Toxicity on the loose

The researchers were guided by computational tools, so they knew that CmdT was likely an ADP-ribosyltransferase due to its similarities to other such enzymes. As the name suggests, the enzyme transfers an ADP ribose onto its target.

To determine if CmdT interacted with any sequences or positions in particular, they tested a mix of short sequences of single-stranded RNA. RNA has four bases: A, U, G, and C, and the evidence points to the enzyme recognizing GA sequences.

The CmdT modification of GA sequences in mRNA blocks their translation. The cessation of creating new proteins aborts the infection, preventing the phage from spreading beyond the host to infect other bacteria.

“Not only is it a new type of bacterial immune system, but the enzyme involved does something that’s never been seen before: the ADP-ribsolyation of mRNA,” Vassallo says.

Although the paper outlines the broad strokes of the anti-phage defense system, it’s unclear how CmdC interacts with the capsid protein, and how the chemical modification of GA sequences prevents translation.

Beyond bacteria

More broadly, exploring anti-phage defense aligns with the Laub Lab’s overall goal of understanding how bacteria function and evolve, but these results may have broader implications beyond bacteria.

Senior author Michael Laub, Salvador E. Luria Professor and Howard Hughes Medical Institute Investigator, says the ADP-ribosyltransferase has homologs in eukaryotes, including human cells. They are not well studied, and not among the Laub Lab’s research topics, but they are known to be up-regulated in response to viral infection.

“There are so many different — and cool — mechanisms by which organisms defend themselves against viral infection,” Laub says. “The notion that there may be some commonality between how bacteria defend themselves and how humans defend themselves is a tantalizing possibility.” 

[wilderun]

Apparently you can add some dog coat color mutations and a gnarly overbite to a grey wolf embryo via CRISPR, plop it in a surrogate hound bitch, and call it a direwolf lol

For real though, the entire “dire wolf brought back from extinction” thing is so wildly misleading as to what they actually did that it’s crazy it picked up as much traction as it has. They have a VERY good media team on board.

These animals are grey wolves with a handful of modifications to their grey wolf genome to give them physical traits that the creators felt resembled dire wolves, based on their research into the partial genome they have available for the species. They did not insert dire wolf genes into these embryos; they merely edited the genes related to size, bone, skull size, and coat color. The coat color they introduced is a dog mutation (recessive red, commonly called “born white” in wolfdogs, is the result of e/e genotype on the E locus).

They’re basically fancy generically engineered wolfdogs — not dire wolves.

[hellsite-proteins]

Are there ways to actually "program" cells to produce these proteins? Like if you fed ribosomes the right RNA sequence, would it synthesize a bullshit protein molecule that does nothing useful, or would it start falling apart immediately in reality? My knowledge on DNA splicing is old and I know near nothing about proteins so I'm just curious if it'd ever be possible to get a real world representation of anything on your blog (setting aside the cost and effort it would take to do that)

yes there is! and you actually got pretty close, but you have to go a bit further back and use DNA (there are ways of delivering RNA to cells, but I haven't done that personally, and for something like protein purification that would not be the method of choice, so I won't be going into that. there are two main ways of inserting coding DNA into cells: using a transient expression plasmid and gene editing. note: all of these will be very brief overviews, so if you want more detail about any part of this, then please ask!

first i'll go over the simpler method which is using plasmids. i've been doing a lot of molecular cloning lately specifically to put proteins into expression vectors so i can purify them and do some work with the pure protein, which is what i assume you would want to do here. (sidetone: once you have some purified protein, there are a number of different things you can do to find out its sequence or structure, but that is a whole other post). plasmids are circular bits of DNA outside of the genome that are not necessary for survival, but carry useful information. most of the ones used in labs will also have an antibiotic resistance gene or some other marker so we can select for only the cells with the plasmid by growing them in media with the right drug. using specific enzymes, we can cut these plasmids and then insert a new fragment of DNA coding for whatever we want, before sealing them back up. Then, a small amount of the plasmid can be transformed into cells (bacteria and yeast are commonly used), which will multiply and make more of your plasmid, as well as whatever it encodes if the conditions are right!

Genome editing is a broad topic, so i'm just going to give you a quick overview of CRISPR-Cas9, which I have a bit of experience with. this isn't something i would do if i just wanted to make a lot of a given protein, but it is useful to look at how much of a protein is made under the native expression system within a cell, or to edit the sequence of a protein being made (and much more...). Cas9 makes a cut in both strands of genomic DNA, but the really neat thing about this is that you can easily tell it specifically where to cut. to do this, you will build a plasmid with the gene for the Cas9 protein, as well as the guide RNA matching the sequence where you want the cut to be. there are other design considerations, but i'll save that for now. once the DNA has been cut, the cell starts to panic and try and stick it back together, but not before some random bases get added and/or deleted from either of the broken ends. this is useful if you want to knock out a gene, because you can go in and mess up its promoter or something similar so that it never gets made. if that is too messy and random, or if you want to knock something in instead of knocking it out, you can also take advantage of the cell's repair mechanisms. if a double stranded break happens, the cell would prefer to fix it properly, and so it will try and remake the DNA using the other matching chromosome as a template. but, if you add in another plasmid instead that contains sequences matching either side of the break with something you want to add in sandwiched in between them, you can make the DNA repair itself with your new sequence!

finally, all of this assumes that the protein would be successfully translated, and would not be so toxic to cells that anything expressing it dies. we would need to add a methionine (start) to the beginning of nearly all of these sequences, and ideally codon optimize them for the specific organism. ordering the custom DNA sequences also wouldn't be cheap, but is also not impossible. an inducible expression system would also help reduce the risk of toxicity and let us make a lot at once if we so desired. we would probably also want to add a tag to make these easier to purify if we wanted to use the protein itself.

again i am not great at being brief and i have no idea how much the average person knows about any of this, so please ask more if anything i've said needs clarification!

letter sequence in this ask matching protein-coding amino acids:

AretherewaystactallyprgramcellstprdcetheseprteinsLikeifyfedrismestherightRNAseqencewlditsynthesieallshitprteinmleclethatdesnthingseflrwlditstartfallingapartimmediatelyinrealityMyknwledgenDNAsplicingisldandIknwnearnthingatprteinssImstcrisifitdeverepssiletgetarealwrldrepresentatinfanythingnyrlgsettingasidethecstandeffrtitwldtaketdthat

protein guy analysis:

you know that feeling when you're writing a test and aren't really sure what the answer is, but you try and put down everything you know in the hopes of getting some part marks? i feel like that's what this protein looks like. there is an assortment of secondary structures all spread out between loops and even a beta sheet, as if AlphaFold was trying its best to make something out of this. still, it doesn't look the way we would expect from an ordered protein, and i do not trust it.

predicted protein structure:

[mindblowingscience]

A diverse set of species, from snails to algae to amoebas, make programmable DNA-cutting enzymes called Fanzors—and a new study from scientists at MIT's McGovern Institute for Brain Research has identified thousands of them. Fanzors are RNA-guided enzymes that can be programmed to cut DNA at specific sites, much like the bacterial enzymes that power the widely used gene-editing system known as CRISPR. The newly recognized diversity of natural Fanzor enzymes, reported Sept. 27 in the journal Science Advances, gives scientists an extensive set of programmable enzymes that might be adapted into new tools for research or medicine. "RNA-guided biology is what lets you make programmable tools that are really easy to use. So the more we can find, the better," says McGovern Fellow Omar Abudayyeh, who led the research with McGovern Fellow Jonathan Gootenberg. CRISPR, an ancient bacterial defense system, has made it clear how useful RNA-guided enzymes can be when they are adapted for use in the lab. CRISPR-based genome editing tools developed by MIT professor and McGovern investigator Feng Zhang, Abudayyeh, Gootenberg, and others have changed the way scientists modify DNA, accelerating research and enabling the development of many experimental gene therapies.

Continue Reading.

[ai-botz]

You:

explain crispr mRNA

Ai:

CRISPR RNA (crRNA) is a sophisticated, programmable molecular tool that leverages the prokaryotic adaptive immune system's mechanism of defense against invasive genetic elements. This RNA-based component serves as a guide to direct the Cas9 nuclease enzyme toward specific genomic loci for targeted editing.

The crRNA structure consists of three distinct regions: the scaffold, the repeat sequence, and the protospacer. The scaffold provides structural support and facilitates binding with the Cas9 protein, while the repeat segment is derived from the CRISPR array in the bacterial genome and promotes crRNA-Cas9 complex formation.

The protospacer region, which constitutes the majority of the crRNA sequence, harbors the desired target specificity information. It possesses a complementarity to a predefined genomic sequence (the target site), thereby enabling the crRNA-guided Cas9 endonuclease to recognize and cleave the target DNA at the precise location.

crRNA synthesis typically involves in vitro transcription from a DNA template or ribosomal RNA processing in vivo. The resulting mature crRNAs are then processed into their functional forms through 5' and 3' trimming, which removes non-essential regions and generates a single-stranded structure that can base-pair with the target site.

Upon binding to Cas9, the crRNA acts as an adaptor, positioning the nuclease at its cognate genomic location. Upon encountering the target sequence, the Cas9-crRNA complex excises the DNA, creating a double-strand break (DSB). The DSB can then be repaired by the cell's endogenous pathways, which may result in precise gene editing via homology-directed repair (HDR) or introduce insertions/deletions (indels) through non-homologous end joining (NHEJ), effectively creating a mutation at the targeted site.

The programmable nature of crRNAs allows researchers to tailor them for specific applications, including gene knockouts, point mutations, and even precise base editing. Moreover, advances in RNA engineering have enabled the development of complex crRNA designs capable of targeting multiple genomic loci simultaneously or recognizing non-canonical target sites.

In summary, CRISPR RNA is a highly specialized, versatile molecular tool that empowers scientists to precisely manipulate genomes through its intricate interactions with Cas proteins and cellular repair machinery. Its unique properties have revolutionized the field of genome engineering, offering unprecedented control over genetic information.

You:

explain how a virus can be used as an mRNA encapsulate to deliver crisper RNA

Ai:

Viruses, particularly those in the families Lentiviridae (e.g., HIV-1), Adenoviridae (e.g., adenovirus serotype 5), and AAVidiae (adeno-associated viruses), can be engineered to function as mRNA encapsulation vectors for CRISPR RNA delivery.

The process typically involves:

Cloning the desired crRNA sequence into an expression plasmid, which is then transfected into virus-producing cells. The viral vector is assembled with its capsid protein and genomic RNA, replacing the original viral genetic material. The modified virus acquires the CRISPR RNA as a passenger payload, encapsulated within its lipid envelope or protein capsid. Upon infection of target host cells, the virus delivers its crRNA cargo into the cytoplasm via endocytosis and lysosomal escape. After entering the nucleus through nuclear pore complexes, the released crRNA can then interact with Cas proteins to initiate genome editing at the desired loci.

The choice of viral vector largely depends on factors such as host specificity, transduction efficiency, and immunogenicity concerns. Lentiviral vectors, for example, are capable of infecting non-dividing cells but may elicit stronger immune responses compared to AAV or adenoviral alternatives. AAVs, being less immunogenic, have become popular choices for gene therapy applications.

Incorporating CRISPR RNA into viral delivery systems offers several advantages over direct crRNA administration. It enables targeted and efficient transduction of cells, enhances the stability of crRNA in circulation, reduces off-target effects due to improved tissue specificity, and can facilitate simultaneous editing or multiplexed gene modifications.

However, challenges persist, including concerns over potential off-target effects from viral-mediated delivery itself, immune responses against the viral vector, and the need for further optimization of viral design and packaging strategies to ensure precise genome editing with minimal collateral damage. Continued research is necessary to refine this approach and maximize its therapeutic potential in various applications, ranging from treating genetic diseases to agricultural biotechnology.

————————————————————————

Virus AI describes as being the likely candidate for spreading crisper RNA

“A digital cryo-electron micrograph of the Adenovirus serotype 5 (Ad-5)/HVR5-33RGD vector. Adenoviruses are non-enveloped, double-stranded DNA viruses that cause cold-like infections of the upper respiratory tract.”

[science-lover33]

Genetic engineering: CRISPR and beyond

In genetic engineering, we find ourselves amidst a scientific revolution with the advent of revolutionary technologies like CRISPR-Cas9. However, our journey into the intricate landscape of genetic manipulation is far from complete. This post delves into the nuanced world of genetic engineering, exploring cutting-edge technologies and their remarkable potential in shaping the future of medicine and biotechnology.

CRISPR-Cas9: Precision at the Molecular Level

CRISPR-Cas9, a revolutionary genome editing tool, stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9. It utilizes a guide RNA (gRNA) to target specific DNA sequences, and the Cas9 protein acts as molecular scissors to cut the DNA at precisely defined locations. This break in the DNA prompts the cell's natural repair machinery to make changes, either through non-homologous end joining (NHEJ) or homology-directed repair (HDR). CRISPR-Cas9's precision allows for gene knockout, modification, or insertion with remarkable accuracy.

Beyond CRISPR: Emerging Technologies

While CRISPR-Cas9 has dominated the field of genetic engineering, numerous promising technologies have emerged on the horizon. These include CRISPR-Cas variants like CRISPR-Cas12 and CRISPR-Cas13, which offer unique advantages such as smaller size, increased specificity, and targeting of RNA. Additionally, base editing techniques, such as adenine base editors (ABEs) and cytosine base editors (CBEs), enable the direct conversion of one DNA base into another without causing double-strand breaks, expanding the range of genetic modifications possible.

Applications in Medicine

The implications of these advancements are profound, particularly in medicine. Genetic engineering can potentially treat various genetic disorders, from cystic fibrosis to sickle cell anemia, by correcting disease-causing mutations at their source. Precision medicine, tailored to an individual's genetic makeup, is becoming increasingly feasible, allowing for personalized therapies with minimal side effects.

Ethical Considerations and Regulation

As we venture further into the genetic frontier, we must acknowledge the ethical considerations surrounding genetic engineering. The ability to modify the human germline, with implications for future generations, raises ethical dilemmas that necessitate rigorous oversight and regulation. The international community is developing guidelines to ensure responsible use of these powerful tools.

Future Directions and Challenges

While genetic engineering offers immense promise, it is not without its challenges. Off-target effects, unintended consequences, and the potential for creating designer babies are among the issues that demand careful consideration. Researchers and ethicists must work in tandem to navigate this uncharted territory.

References

Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096.

Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., … & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157.

Kime, E. (2021). CRISPR and the ethics of gene editing. Nature Reviews Genetics, 22(1), 3-4.

This post only scratches the surface of the profound transformations occurring in genetic engineering. The relentless pursuit of knowledge and ethical exploration will shape the future of this field as we continue to unlock the intricate secrets of our genetic code.

[darkmaga-returns]

If you stand on a river bank and wait for failed gene therapies to float by… you will be flooded! Admission of failure from the pharma propaganda outlets, on the other hand, is a rare event. This washed up in my inbox recently:

“Exclusive: CRISPR delivery startup, based on UC Berkeley and UCSF science, shuts down.”

and, an even more damning long article questioning the future of the entire CRISPR segment:

“Will CRISPR matter?”

I am posting some excerpts here, with my commentary.

First, what is CRISPR? It is a Nobel Prize winning (of course) “gene editing” technology, long hyped-up as a revolution in health and medicine, and advertised as absolutely 100% functioning and real method for making genetically modified organisms (GMOs) of animals, plants and even humans. Wikipedia says:

The technique is considered highly significant in biotechnology and medicine as it enables editing genomes in vivo and is precise, cost-effective, and efficient. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also offers potential in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer. However, its use in human germline genetic modification is highly controversial. The development of this technique earned Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in Chemistry in 2020.

They even put someone in China in jail for reportedly making GMO designer babies with this technique. So, it must work, right? If you can make an entire designer baby, or cows without horns, then you should be able to fix someone’s vision with this method, i.e. fix just one feature instead of the whole baby! Turns out, no. You can’t. Also, the hornless cows were a major screw up (MIT Review in 2019). And, it turns out that when scientific proof is required for CRISPR claims in drug development (not in liability- and regulation-free vaccines) it resolutely fails!

[quasisnipr1048]

On the Woolly Mouse, Dire Wolves, Gene Editing, and Conservation

I found it kind of comical that within my own circles, I generally had the same initial reaction to the work of Colossal Biosciences. After sitting with it for a while and really thinking about it, I don't believe my thoughts on the matter have changed much.

Let's start with the Woolly Mouse. Everyone, and I mean everyone, seemed to love them. It was novel, it was unusual. And they looked adorable. Except, I felt more concern than I did amusement. If you've followed me for any length of time, you are probably aware that I am an engineer (it does say it in my title card). I've worked with safety critical systems before where if something seems wrong, it's probably wrong and you need to figure out why and fast. The Woolly Mouse seemed... wrong. It felt wrong. At the time, I didn't follow the story very closely because it just seemed so strange. And in this day and age, I thought it was manipulated or generated by AI. I ignored it.

And then, this week, another gene research story. Some company "brought back" Dire Wolves, a species that's been extinct for the past 10,000 years.

Something felt off again, and this time, I felt it much more strongly. Colossal Biosciences was named again. Who the hell are these people and why are they doing this? They are the same people who made CRISPR, but with a hell of a lot of funding behind them, and they claim they want to preserve endangered species with the power of gene editing.

And I just have to ask: Why?

Not, "Why are you wanting to preserve endangered species?" No, "Why do you think gene editing is the answer?"

I think my biggest criticism of it is the same criticism echoed by Ian Malcolm in Michael Crichton's Jurassic Park: The scientists were so preoccupied with whether or not they could that they didn't stop to think if they should. By their own words, Colossal is working to "de-extinct" animals, which, I will agree, might be a noble cause. But let's say they do succeed in "de-extincting" these animals and consider these three scenarios.

Maybe everything goes according to plan. This company is actually responsible with the power they wield and actively participates in conservation efforts for endangered animals. They help save hundreds of species with gene editing. This is idealistic but a good outcome.

Maybe gene editing in this manner does not produce good results in the long term. These revived species have a hard time reproducing and adapting to the way the world is. We are able to study them for a time before they eventually go extinct again. As harsh as this sounds, I also consider this a good outcome, if unethical. We should not revive a species just for them to go extinct again.

Maybe everything goes according to plan, but the company is not as responsible with the power they wield. Some of these revived species escape captivity a la Jurassic Park and start reproducing, either with each other or with the base animal they were created from (see Dire Wolves). Suddenly we have an invasive species on our hands and no proper controls in place to handle them. This should be obvious, but this a very bad outcome.

So, if it isn't clear yet, I'm very much on the fence about using gene editing as a means of conservation. I think there's biological and ecological implications that have not been considered, and yet, the research marches forward behind the pockets of venture capital. I find it to be a slap in the face of ongoing conservation efforts that there are those who would rather use gene editing and ignore its implications to "save the planet". I get it; we as a species are in a unique position to intervene and correct our mistakes. But trading one villain for another when it comes to ecological collapse is not the solution.

(I feel the need to inject this side tangent that for the supposed "Dire Wolves" they only changed 20 spots in their genome, so I tend to agree with the scientist that said Colossal just made genetically modified grey wolves. My main question then is does this count and conservation or are we just creating some chimera in a lab. This does bring up other questions about biology and genetics that I don't think I'm even qualified to ask.)

At best, maybe gene editing our way out of an ecological collapse does work and I'm proven wrong. I would love to be wrong. I'm an engineer, not a biologist. I don't fully understand what all goes into this science. Maybe I missed something. But, I'd like to think a good engineer knows when to listen to their gut and double check that everything is in order. And my gut has a very bad feeling about this. Gene editing, to me, is right up there with nuclear power. It shouldn't be feared, but it commands respect.

That being said, I don't want to be around when the Oppenheimer of gene editing comes along to become destroyer of worlds.

[biopractify]

🧬 How Bioinformatics is Revolutionizing Pharma & Drug Discovery 🚀

🔬 Bioinformatics is changing the way we develop medicines. From AI-driven drug discovery to genomics-based precision medicine, biotech is making healthcare smarter, faster, and more personalized. But how exactly is it transforming the pharma industry? Let’s explore!

📖 Keep scrolling to uncover how AI, data science, and bioinformatics are shaping the future of medicine!

✨ The Impact of Bioinformatics in Drug Discovery

🔹 1. Finding the Right Drug Targets Faster

📌 Old Way: Scientists spent years identifying potential drug targets. 🚀 Now: AI & bioinformatics analyze vast genomic datasets to find disease-related genes in hours!

💡 Example: AI-driven analysis helped identify key targets for cancer immunotherapy drugs like Keytruda (pembrolizumab).

🔹 2. Computational Drug Design & AI-Powered Screening

💊 Why waste years testing thousands of compounds?

Bioinformatics speeds up drug discovery through virtual screening & molecular simulations.

AI models predict how different drugs will interact with the body.

🔥 Pfizer used AI-driven bioinformatics to speed up the development of COVID-19 antivirals!

🔹 3. Personalized Medicine & Biomarker Discovery

✨ Not all treatments work for everyone. Bioinformatics makes medicine more precise!

Identifies genetic markers for disease risk.

Develops customized treatments for cancer & genetic disorders.

Predicts how patients will respond to a drug before prescribing it.

💡 Example: The breast cancer drug Herceptin was developed using bioinformatics to target HER2-positive patients.

🔹 4. CRISPR Gene Editing & Next-Gen Therapies

🧬 Bioinformatics guides CRISPR gene editing, helping scientists:

Design gene therapies for rare genetic disorders.

Reduce off-target mutations for safer treatments.

Improve DNA sequencing for precision medicine.

🔥 Companies like Intellia Therapeutics are using AI-powered bioinformatics for gene editing!

🔹 5. AI-Optimized Clinical Trials & Drug Repurposing

👩‍⚕️ Clinical trials cost billions. Bioinformatics helps optimize them!

Predicts side effects using AI.

Finds new uses for existing drugs (like how Remdesivir was repurposed for COVID-19).

Helps pharma companies save time & money while improving success rates.

💡 Example: AI-driven bioinformatics helped repurpose existing drugs for rare diseases!

🚀 The Future of Bioinformatics in Pharma

💡 The biotech revolution is just getting started! Expect to see: ✅ AI-powered drug design in real time. ✅ Nanomedicine & regenerative therapies. ✅ Affordable personalized medicine.

✨ What excites you most about the future of bioinformatics? Drop your thoughts in the comments! 💬👇

[bpod-bpod]

Editing the Future

Jennifer Doudna – born on this day (19th February)– shared the 2020 Nobel Prize for Chemistry with Emmanuelle Charpentier for developing a genetic engineering technique called CRISPR-Cas9. Based on a naturally occurring defence system used by bacteria to expunge foreign DNA from their genome, CRISPR-Cas9 has revolutionised both biomedical and plant research readily revealing the impact of editing genes in living cells and model organisms, and is being applied in human genome editing to correct disease-causing gene faults and deliver gene therapies

Image by Christopher Michel, on Flickr

Image originally published with a Creative Commons Attribution 2.0 Generic (CC BY 2.0)

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[anarchistfrogposting]

CRISPR, arguably the most fundamental advancement in genetic editing since the Human Genome Project, was discovered because one guy found some weird sequences in a random species of bacteria found in salt geysers, and two other dudes figured out they arose to protect it from viruses. When you look at the history of most scientific advancements, about 95% of them are based on an adaption or enhancement one seemingly low-impact research paper made a few years prior. It is fundamentally collaborative and cumulative.

Culture is so obsessed with the idea of lone geniuses that it doesn't really appreciate that most of the progress of science (and likely every other discipline) occurs collaboratively, in babysteps, and usually through a lot very tedious, utterly unsexy, work.

[anujmrfr]

Beyond Lifespan: How Are Scientific Breakthroughs Redefining Human Longevity?

What Is the Core Focus of Longevity Technology?

The "longevity market" refers to a rapidly emerging and transformative sector focused on extending not just human lifespan, but more importantly, healthspan – the period of life spent in good health, free from chronic disease and disability. This multidisciplinary field integrates cutting-edge advancements across biotechnology, pharmaceuticals, digital health, regenerative medicine, and lifestyle interventions. Its core aim is to understand, prevent, and even reverse the biological processes of aging, thereby improving quality of life in later years and reducing the burden of age-related diseases. Driven by an aging global population and increasing scientific understanding of aging mechanisms, longevity technology represents a paradigm shift from treating diseases reactively to proactively managing the aging process itself.

What Scientific Breakthroughs Are Powering Longevity Innovations?

The rapid growth in longevity technology is fueled by groundbreaking scientific discoveries and technological innovations across several key areas:

Cellular and Molecular Therapies: Research into cellular senescence (the accumulation of "zombie cells" that contribute to aging and inflammation) has led to the development of senolytics, a new class of therapeutics designed to selectively remove these senescent cells. Early studies show promise in ameliorating age-related conditions. Similarly, research into telomere extension therapies and stem cell treatments aims to rejuvenate tissues and organs, addressing age-related degeneration at a fundamental cellular level. Genetic editing technologies like CRISPR are also being explored for their potential to correct age-related genetic predispositions.

Genomics and Epigenetics: Advances in preventive genomics and epigenetic clocks are revolutionizing our understanding of biological aging. Genetic testing can identify individual predispositions to age-related diseases, allowing for personalized interventions. Epigenetic clocks measure an individual's biological age, which may differ from their chronological age, providing a more accurate assessment of aging and the effectiveness of anti-aging interventions. This data-driven approach enables highly personalized longevity strategies.

AI and Digital Health Platforms: Artificial intelligence (AI) and machine learning (ML) are becoming indispensable tools in longevity research and application. AI algorithms can analyze vast datasets of biological and health information to identify biomarkers of aging, predict disease risks, and accelerate the discovery of new drugs targeting age-related conditions. AI-powered platforms are also being developed for personalized health recommendations, biomarker monitoring, and tailored longevity interventions based on an individual's genetic, metabolic, and lifestyle data. Wearable devices and fitness trackers integrated with AI platforms allow for real-time tracking of vital health metrics, enabling proactive health management and early detection of potential issues.

What Are the Key Applications and Future Trajectories of Longevity Technology?

The applications of longevity technology are diverse and expanding rapidly. In regenerative medicine, stem cell therapy and tissue engineering are being explored to restore damaged tissues, rejuvenate organs, and potentially reverse age-related degeneration in areas like skin, muscles, and joints. Longevity-focused nutrition and supplements, including nutraceuticals and anti-aging compounds like NAD+ boosters, resveratrol, and rapamycin-based compounds, are a significant part of the consumer-facing longevity market, aiming to optimize cellular health and mitochondrial function.

Beyond direct medical interventions, the longevity sector is also influencing broader societal changes. There's a growing focus on age-friendly urban planning, with communities redesigning public spaces and infrastructure to support older residents. Workforce policies are adapting to accommodate aging workers, with an emphasis on reskilling and upskilling initiatives.

The future of longevity technology is incredibly promising. Research will continue to delve deeper into the fundamental mechanisms of aging, leading to the development of more targeted and effective interventions. The integration of multi-omics data (genomics, proteomics, metabolomics) with AI will provide even more comprehensive insights into individual aging profiles. The drive towards accessible and affordable longevity solutions will likely see a proliferation of at-home diagnostic kits and personalized digital health platforms. As scientific understanding advances and investment surges, longevity technology holds the potential to fundamentally reshape human health, extending not just how long we live, but how well we live throughout our lives.

Contact:

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+1 628 258 0071(US)

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Website: https://www.marketresearchfuture.com

[vijaysethupati]

Revolutionizing Clinical Research with Genomic Personalization

In the ever-evolving world of healthcare, clinical research stands as the backbone of medical advancements. The latest breakthrough in this realm is genomic personalization—a transformative approach that tailors treatment based on individual genetic makeup. This advancement isn’t just enhancing patient outcomes; it’s also changing how clinical research is conducted, learned, and implemented across the globe.

Whether you're exploring clinical research coaching with 100% placement, wondering what exactly is clinical research, or curious to know is clinical research a good career in India, this article will unfold answers while highlighting the crucial role genomics plays today.

Understanding Genomic Personalization in Clinical Research

Genomic personalization refers to leveraging an individual's DNA information to develop customized therapies. Instead of the "one-size-fits-all" treatment model, it focuses on precision medicine—ensuring that each patient receives treatments best suited to their genetic structure. This new wave in clinical research is making studies more targeted, efficient, and meaningful.

Incorporating genomics allows researchers to:

Identify high-risk individuals early.

Predict how patients will respond to specific drugs.

Eliminate trial-and-error approaches in treatment.

Reduce adverse drug reactions.

These benefits make genomic personalization a cornerstone of modern clinical trials, thus raising the need for skilled professionals who understand this innovation deeply.

What Exactly is Clinical Research?

Before diving further, let’s clarify what exactly is clinical research. Clinical research involves studies and trials on human participants to evaluate medical, surgical, or behavioral interventions. It's essential for developing new treatments, medications, diagnostic tools, and understanding disease progression.

There are typically two types:

Observational studies – where researchers observe patients without altering their treatment.

Clinical trials – where new interventions are tested for effectiveness and safety.

With genomics in the mix, both these types are becoming increasingly data-driven and specific to individual biology.

Why Are Clinical Trials Essential?

With the rise of genetic science, the question why are clinical trials essential becomes even more significant. Clinical trials now go beyond testing new drugs. They validate targeted therapies derived from genomic research. These trials ensure that:

New drugs are safe and effective.

Therapies are tested across diverse genetic groups.

Innovations like CRISPR and gene editing are applied responsibly.

In short, clinical trials ensure the integration of new genomic techniques into everyday medicine, making them indispensable in today’s healthcare ecosystem.

Career Scope: Is Clinical Research a Good Career in India?

Absolutely, clinical research is a good career in India. As the global pharmaceutical and biotech industries grow, India is emerging as a major hub due to its vast patient pool and cost-effective operations. Additionally, the adoption of technologies like genomic personalization is opening high-paying and impactful roles in research, data analysis, and medical innovation.

In fact, numerous institutions now offer clinical research certification with internships to bridge the skill gap. These programs not only provide theoretical knowledge but also offer hands-on experience with real-world genomic tools and trial methodologies.

Pathway to Success: CRC Training and Certification in India

A Clinical Research Coordinator (CRC) plays a crucial role in managing trials efficiently. To meet industry demands, many institutes offer CRC training and certification in India. These courses are designed to cover:

Ethical considerations in trials

Data collection and documentation

Genomic data interpretation

Patient recruitment and engagement

Such training empowers students and professionals to adapt to personalized medicine trends and enhances their employability.

Advantages of Clinical Research Coaching with 100% Placement

If you're planning to build a future in this field, opting for clinical research coaching with 100% placement can be your game-changer. Reputed institutes not only train candidates in theory and practicals but also ensure they get placed in leading pharma, biotech, or healthcare companies.

Benefits include:

Practical exposure to genomic tools

Experience in trial setup and documentation

Access to expert mentors and alumni networks

Guaranteed entry-level jobs in top CROs and hospitals

Conclusion

The fusion of genomic science with clinical research is not just a scientific revolution—it’s a career revolution. From personalized cancer therapies to targeted drugs for rare diseases, the scope is boundless. For students and professionals alike, now is the best time to step into this dynamic field.

Whether you're taking up clinical research certification with internships, pursuing CRC training and certification in India, or seeking clinical research coaching with 100% placement, ensure your learning includes exposure to genomics. Because in the coming years, precision medicine won’t be the future—it will be the norm.

So, are you ready to be part of the next healthcare transformation?

[geneubi]

Beyond the Single Base: Understanding Point Mutation Cell Lines in Modern Biomedical Research

In the age of genomic precision, even a single nucleotide can determine the fate of a cell—whether it thrives, fails, or transforms into a tumor. Point mutation cell lines, which carry targeted single-base changes in their DNA, are now widely used as functional research models in molecular biology, disease modeling, and drug development.

This article offers a comprehensive overview of what point mutations are, how they are engineered into cell lines, the technologies involved, and why they matter more than ever in translational and basic science.

What Are Point Mutations—and Why Do They Matter?

A point mutation involves the alteration of a single nucleotide base in the DNA sequence. Depending on where this change occurs and what it encodes, the effects can range from completely silent to disease-causing. Key types include:

Missense mutations: A single base change leads to a different amino acid, potentially altering protein function.

Nonsense mutations: A change introduces a premature stop codon, often leading to truncated, nonfunctional proteins.

Silent mutations: No change in amino acid, but potential effects on splicing or translation efficiency.

Splice site mutations: Disrupt exon-intron boundaries, affecting mRNA processing.

Regulatory region mutations: Alter gene expression levels rather than protein structure.

Understanding these mutations is essential in diseases like cancer, neurodegenerative disorders, and inherited syndromes. Studying them in human or animal cells requires precise gene editing and stable model systems—hence the rise of point mutation cell lines.

How Are Point Mutations Introduced into Cell Lines?

Modern genome editing technologies have made it possible to recreate disease-relevant mutations in cell lines with high precision. The most widely used method is based on CRISPR-Cas9 combined with homology-directed repair (HDR).

Key Steps:

Targeting: A single guide RNA (sgRNA) directs the Cas9 enzyme to a specific DNA sequence, creating a double-strand break.

Template Introduction: A synthetic DNA donor (usually ssODN) carrying the desired mutation is introduced.

Repair and Integration: The cell's natural repair machinery uses the donor as a template, incorporating the mutation during the S or G2 phase.

This strategy allows researchers to create either heterozygous or homozygous mutant clones, depending on the study design.

Enhancing Efficiency: Overcoming the HDR Bottleneck

One of the main challenges in point mutation modeling is the low efficiency of HDR. Many cells prefer the faster, error-prone NHEJ pathway, making precise edits rare without intervention.

To address this, various labs have developed strategies to enhance HDR, including:

Chemical inhibitors of NHEJ (e.g., SCR7, DNA-PK inhibitors)

Cell cycle synchronization

Donor template optimization

Engineered Cas9 variants with increased HDR bias

Among notable advancements is EZ-HRex™, a proprietary system that integrates CRISPR with a small molecule enhancer (U+), significantly improving HDR rates in multiple cell types. Though initially developed for internal use, platforms like EZ-HRex have influenced how commercial and academic labs approach mutation modeling today.

CRISPR vs. RNAi: Why Editing Wins for Point Mutation Research

While RNA interference (RNAi) can transiently knock down gene expression, it doesn’t alter the genome. For researchers aiming to explore the functional impact of specific mutations, CRISPR-Cas9 remains the preferred tool because:

It creates stable, inheritable edits

It can precisely reproduce known pathogenic variants

It allows control over zygosity (heterozygous vs. homozygous)

For studying long-term cellular behavior, signaling cascades, or drug response based on specific base changes, RNAi simply falls short.

Applications of Point Mutation Cell Lines

Engineered cell lines carrying point mutations have opened up new research avenues in:

1. Cancer Biology

Mutations in TP53, KRAS, EGFR, and IDH1 are frequently modeled to study their role in oncogenesis, therapy resistance, and cell cycle control.

2. Rare Disease Modeling

Point mutations linked to diseases like cystic fibrosis (CFTR gene) or Marfan syndrome (FBN1 gene) are replicated in vitro to study disease mechanisms or test gene correction approaches.

3. Functional Genomics

Point mutation models help clarify structure-function relationships in proteins, such as ATP binding sites or phosphorylation motifs.

4. Drug Screening and Target Validation

When a mutation alters drug sensitivity (e.g., EGFR T790M in lung cancer), corresponding cell lines allow pharmaceutical testing in a genetically matched system.

Detection: How Are Edits Verified?

Validating a single-base change requires sensitive and accurate tools. Common approaches include:

Sanger sequencing: Still the most widely used method for confirming precise edits.

qPCR and allele-specific PCR: Useful for clone screening in large experiments.

Next-generation sequencing (NGS): Detects on-target and off-target events at genome-wide resolution.

Droplet Digital PCR (ddPCR): Ideal for detecting rare or mosaic mutations.

Restriction fragment analysis: If the mutation affects an enzyme recognition site.

Post-editing validation may also involve functional assays, protein expression analysis, or phenotypic observation depending on the target gene.

Case Insight: TP53 R175H Mutation

TP53 is one of the most frequently mutated genes in human cancer. A well-known example is the R175H mutation, which alters the tumor suppressor’s function. In landmark experiments, researchers introduced this mutation into colon cancer cell lines and observed not just loss of function, but gain-of-function activity, including altered chromatin regulation and promotion of tumorigenesis.

Such findings underscore the value of point mutation models—not just for mimicking disease, but for uncovering previously unknown functions of mutated genes.

Are All Point Mutations Harmful?

Interestingly, not all point mutations have negative consequences. Some are neutral, others are even adaptive. It’s the context—both genetic and environmental—that determines pathogenicity.

However, when a mutation hits a critical domain in a gene involved in DNA repair, apoptosis, or cell signaling, it can have a profound effect. That’s where point mutation models become invaluable: they provide a direct window into the causal effects of a single genetic change.

Final Thoughts: A Single Base, A World of Insight

As we continue to explore the genetic underpinnings of disease, point mutation cell lines are proving to be more than just a research tool—they're foundational to functional genomics, precision medicine, and therapeutic discovery. The ability to faithfully reproduce and study even a single base change in a controlled cellular environment offers researchers a level of insight that was once unimaginable.

Whether you're exploring a cancer driver mutation or modeling a rare inherited disorder, these models are shaping the future of how we understand—and treat—genetic disease.