Stanford Mini CRISPR Improves Gene Editing Inside Cells

Bioengineers have repurposed a “non-working” CRISPR system to make a smaller version of the genome engineering tool. Its smaller size should make it easier to deliver into human cells, tissues and the body for gene therapy.

“CRISPR can be as simple as a cutter, or more advanced as a regulator, an editor, a labeler or imager. Many applications are emerging from this exciting field,” said Stanley Qi, who is also an assistant professor of chemical and systems biology in the Stanford School of Medicine and a Stanford ChEM-H institute scholar.

In a paper published Sept. 3 in Molecular Cell, Qi and his collaborators announce what they believe is a major step forward for CRISPR: An efficient, multi-purpose, mini CRISPR system. Whereas the commonly used CRISPR systems – with names like Cas9 and Cas12a denoting various versions of CRISPR-associated (Cas) proteins – are made of about 1000 to 1500 amino acids, their “CasMINI” has 529.

The researchers confirmed in experiments that CasMINI could delete, activate and edit genetic code just like its beefier counterparts. Its smaller size means it should be easier to deliver into human cells and the human body, making it a potential tool for treating diverse ailments, including eye disease, organ degeneration and genetic diseases generally.

To make the system as small as possible, the researchers decided to start with the CRISPR protein Cas12f (also known as Cas14), because it contains only about 400 to 700 amino acids. However, like other CRISPR proteins, Cas12f naturally originates from Archaea – single-celled organisms – which means it is not well-suited to mammalian cells, let alone human cells or bodies. Only a few CRISPR proteins are known to work in mammalian cells without modification. Unfortunately, CAS12f is not one of them. This makes it an enticing challenge for bioengineers like Qi.

“We thought, ‘Okay, millions of years of evolution have not been able to turn this CRISPR system into something that functions in the human body. Can we change that in just one or two years?’” said Qi. “To my knowledge, we have, for the first time, turned a nonworking CRISPR into a working one.”

Indeed, Xiaoshu Xu, a postdoctoral scholar in the Qi lab and lead author of the paper, saw no activity of the natural Cas12f in human cells. Xu and Qi hypothesized that the issue was that human genome DNA is more complicated and less accessible than microbial DNA, making it hard for Cas12f to find its target in cells. By looking at the computationally predicted structure of the Cas12f system, she carefully chose about 40 mutations in the protein that could potentially bypass this limitation and established a pipeline for testing many protein variants at a time. A working variant would, in theory, turn a human cell green by activating green fluorescent protein (GFP) in its genome.

“At first, this system did not work at all for a year,” Xu said. “But after iterations of bioengineering, we saw some engineered proteins start to turn on, like magic. It made us really appreciate the power of synthetic biology and bioengineering.”

The first successful results were modest, but they excited Xu and encouraged her to push forward because it meant the system worked. Over many additional iterations, she was able to further improve the protein’s performance. “We started with seeing only two cells showing a green signal, and now after engineering, almost every cell is green under the microscope,” Xu said.

“At some moment, I had to stop her,” recalled Qi. “I said ‘That’s good for now. You’ve made a pretty good system. We should think about how this molecule can be used for applications.’”

In addition to protein engineering, the researchers also engineered the RNA that guides the Cas protein to its target DNA. Modifications to both components were crucial to making the CasMINI system work in human cells. They tested CasMINI’s ability to delete and edit genes in lab-based human cells, including genes related to HIV infection, anti-tumor immune response and anemia. It worked on almost every gene they tested, with robust responses in several.

Molecular Cell – Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Highlights
• Protein and RNA engineering enable Cas12f to function robustly in mammalian cells

• The engineered CasMINI is compact and less than half the size of Cas9 and Cas12a

• CasMINI is efficient and specific for gene activation and is comparable with Cas12a

• CasMINI is versatile and allows robust genome editing and base editing

Summary
Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

SOURCES- Stanford, Molecular cell
Written by Brian Wang, Nextbigfuture.com

39 thoughts on “Stanford Mini CRISPR Improves Gene Editing Inside Cells”

  1. "tend to think that all selection is a positive thing making us healthier and a better fit". That is only true of the really important stuff, that will kill fast if done wrong. Most is sex stuff, entirely irrational from "fitness" as Darwin defined. Then, throw in human infantile needs and crazy stuff like sailing.

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  2. There are a handful of genes that were broken in the past that would be useful today in humans. There was a site with a list, but I can't find it. One is the gene for vitamin C production. Evidently, some time in the past all humans/ancestors of humans got enough vitamin C from their diet to gain no benefit from producing it in their bodies. So when it broke, that was a modest advantage as that is one less thing for the body to expend energy making. Before long, no one had a working gene for vitamin C. We still have the gene. They know where it is…it just does not function.
    Between 1500 and 1800 over 2 million sailors died of scurvy (caused by lack of vitamin C). Every time they crossed the ocean a lot of sailors died. More dangerous by far than space travel today. The vast majority of animals make vitamin C. There is not much in the tissue because it is made as needed…so you don't get much from eating meat even if you ate it raw.
    Broken genes can be active too, just making the wrong thing. Perhaps 80% of human cancers may be facilitated by just such a gene: https://medium.com/predict/the-broken-gene-that-promotes-advanced-cancer-de9175f99bbe
    We also tend to think that all selection is a positive thing making us healthier and a better fit. I think that often a change is harmful long term and these changes are more like the scaring of a species. I think sickle cell is just such a change. And when malaria is defeated, it will be obviously bad.

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  3. This is very similar and closely related in actuality to the old "Nature v Nurture" dispute. "is not needed" if other mechanisms do the job. Needed now, in other species, if that is the way the evolution happened. "Not needed" if intelligent design, could have done it right everywhere. Present in most cases. That there are existing important uses of the junk is all that is needed to say it can be important. Some Nurture effect, esp the reproduced epigenetic stuff, disproves the Nature claim, that there is NO such effect. The claims are not symmetric, Nurture is an easy case to prove.

    Now, this is not about the safety of CRISPR, but rather the idea that there are long feedback loops that help with the epigenetic function, where Nurture input determines permanent changes in gene expression. Patterns to match the Nurture input and thus inform the command of the epigenetic changes must be somewhere. They could be reversed for some reason. It may be that epigenetic changes are the way genes first started working, as the RNA was less well developed.

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  4. There was an off-the-cuff remark that the IQ required to destroy the world by yourself seems to be dropping by about 1 point per 18 months.

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  5. Biotech capable of killing millions will be within technical reach of loners in their bunkers(basements) within the decade.

    May you live in interesting times.

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  6. I expect that, had we a sampling of hundreds, thousands, or millions of species that had reached our current levels, and then continued, we would find they used intelligence to continue to evolve (to something “better”) where blind evolutionary processes would almost certainly never have taken them in the time available. 

    In a sense, it seems possible, even likely, that intelligence is a tool to allow advancement to superior forms, and ubiquitous enough (had we enough samples) for us to say it is part of the natural process of evolution.

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  7. That did not look objective to me. There is a lot of irrational human worship that taints various areas of research. This is one, another is brain research.
    The very fact that people would think that "junk" is pejorative at all rather than descriptive, and requiring defense, shows the bias.
    As I said, noncoding regions can have important functions. But the facts are that some of the stuff that is active is destructive…aging and disease causing. Much of it wasteful, using resources for things that have no role and can slow important processes with clutter. And some does not do a thing.
    Any DNA that is important does not very much between people or there are a set number of variants that 99%+ of people have. If there are hundreds of versions randomly changed, then it is clearly useless. Every person has around 100 new errors…not present in their parents. This breaks stuff but only a small percentage of the DNA is critical, so errors are usually no big deal. When they are critical, the embryo may not be viable. It may not get past the single cell, if some genes are damaged. 
    Researchers know all this, so they look at what is conserved, what isn't, we don't need.
    There are species that actively remove junk from their DNA (a few plants). As a result they have very concise genomes.
    I think this demonstrates better than any experiment, that most of this is not needed: http://www.buffalo.edu/news/releases/2013/05/023.html

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  8. The question of side edits in the junk may soon be helped as we actually do get full copy of everything. At least we will know what is edited. But the overall questions remain as we still don't know what these things do. So, who decides?

    BEIJING (AP)
    Chinese leaders want to “direct the constructive energies of all
    people in one laser-focused direction selected by the party,” Andrew
    Nathan, a Chinese politics specialist at Columbia University, said in an
    email.

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  9. I am reminded of the controversy surrounding the use of embryonic stem cells in research; I've heard more than one Doctor of Philosophy or of Medicine stating categorically that, unless we consented to scavenge those human beings for parts, then we would be condemning the field to stagnation and preventing the development of treatments that could do so much good to so many people… And as the pluripotent techniques were developed, those same doctors quietly pretended they had said nothing in the first.

    We must be careful with the dichotomies we insert ourselves into. Oftentimes they only seem like dichotomies because we are, as a species, extremely myopic. I am not saying I am categorically opposed to genetic manipulation; I am saying that CRISPR isn't good enough a tool to be practically used. Maybe some other mechanism might more reliably implement the changes that are planned on the target genome with an acceptable level of side effects, but if we accept CRISPR as "good enough" it might never come.

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  10. The article describes a *junk* aided balance between long life and cancer. More tele means both longer life but also more cancer. Reminds me of the mice that poison their food with aflatoxin so it will kill other animals that eat their food, but the mice don't live long enuf to die from it. Humans seem to have a system that kills off the powerful/lazy overconsumers by making them fat. Ideas that change within a short time are pretty much unique to humans, so the Planck observation is about *young* people who won't change. You have to wait for them to get old and die before things change. It is true. I have been talking Primal Science and High Frontier for over 40 years. It is TRUE.

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  11. Cells in the body are programmed to commit harakiri when they are no longer functioning properly – too many breaks in the DNA, nonfunctioning mitochondria, maybe even just unused muscle. Individual people within society act the same way – when their usefulness ends, they go gentle into that good night. Huge numbers of stubbornly surviving geriatrics, full of outmoded ideas and attitudes, would be as toxic to the body politic as a cancerous growth. As Planck quipped, 'Science advances one funeral at a time.'

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  12. If we really want a Final Solution to the Car Problem, it might be simpler just to make giant armadillos than to reach all the way back for dinos. Maybe big enough to get rolled a few metres, while flattening the front of offending vehicle and setting off all the airbags.

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  13. Sadly, we have no idea how many pieces are required to achieve longevity. If we are going to think of it as a jigsaw puzzle, then it's one we've found with all the bits scattered over the ground in the wilderness.

    • We have no idea how many pieces there are
    • We have little idea of what the final picture is supposed to look like
    • We don't know how far we'll need to search to find all the pieces. Metres? Km? 100 km?
    • We don't know if all the pieces are even here on the ground, some may have been washed into streams and be at the bottom of the ocean, some may have been destroyed and be totally irretrievable.
    • We could be attacked by bears while we are looking.

    We have started to click some of the bits together. It seems like it is a 2D jigsaw, not a 3D (or 7D??) monstrosity. We can make out what looks like some shapes we recognise in the few assembled bits.

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  14. "that given the right conditions, the body will heal itself." While I agree with his overall message, the "greatest healer" is clearly Janov. Unless your guy published the cure for Neurosis prior to 1970. "Cancer and heart disease (I would add suicide) are not the major killers they are thought to be. Neurosis is" – Janov Neurosis also causes one to partake of many of the *bad* foods you describe. Janov claims to be the first to state the painkilling nature of food, with specific and accurate notion of Pain. Curiously, one of the only actual descriptions of Janov to come out in the articles concerning Lennon long ago described a scene at the end of the article. In it, the *cult guru* being described, Janov, stated something like "Not only have we found the cure for mental illness, but for most disease as well." The followers nodded in agreement. This is stated a proof that Janov is a quack. It would be very convincing proof, but for one fact. "Not only have we found the cure for mental illness, but for most disease as well." is TRUE!

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  15. In these sort of *extreme* philosophical , ethical or moral situations, you are almost certain to get no satisfactory simple rule, but also, you have to get past "informed" in informed consent before getting to the topic, as that is a standard problem. I think more of the whole family (name??) that tested chemicals by tasting, etc, knowing of the danger. "Suffer" was their family motto. There are other important things people believe that are false that we allow. They do/don't believe in the one true gods, for example. Particularly if they disagree with us, we should consider the bottom line rights.

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  16. I see where you are coming from, but I still think it is a kind of moral grey area. If it was just laying dinosaur eggs, it would be fine, but I fear that most people who consented to such kinds of emergency treatments would do so under a misapprehension of the real stakes here, thinking that their actual chances of cure are much much higher than they actually are, while not really understanding that in the vast majority of cases they would simply die regardless, but possibly in quite a bit more pain than they would otherwise.

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  17. He has finally sorted out the food we need that is beneficial for us, after all the bought research financed by the bad food industry, he also sorted out bad conventional medicine caged by misled dogma from good one, all in the name of science. Most importantly, he has found the most fundamental cause of all disease and established the most important principle in medicine, alternative or dogma, that given the right conditions, the body will heal itself. I have experienced it first hand. Thus, he deserves this title. You better delve into his knowledge before starting calling people names.

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  18. "Medical Medium, the greatest healer that ever lived,"
    That sort of extreme praise goes a long way toward convincing me that he & you are cranks.

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  19. The catch is that, while crispr is good enough for in vitro genetic engineering, and you might even risk it in vivo if you were otherwise facing a short term death sentence, (Wolfram syndrome, perhaps.) it's not really good enough for in vivo application for something less urgent. Some fraction of the time it inserts in the wrong place, and if used whole body you'd almost certainly end up with some cancers, or something like that.

    We need better genetic engineering for in vivo use. I really think we need to concentrate on delivery mechanisms for artificial chromosomes.

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  20. Just found recent Quanta (*good* imho) overview. I see several cases of epigenetic or similar activity listed, control the genes even without being them. They argue that the word *junk* is too pejorative. My only useful thot may be that life is seemingly always trying to get bigger and this sort of thing is tolerated for that end only. Harder to swallow?

    https://www.quantamagazine.org/the-complex-truth-about-junk-dna-20210901/

    edit:
    https://cropforlife.com/junk-dna/

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  21. The reason some of it matches is because the regular gene was duplicated and put in backwards. Even modified, it won't work because it is backwards. There are many errors of this type. And it is just junk.
    That is not to say that none of the noncoding stuff has function, just not this reversed stuff.
    Viruses also inserted stuff in the past naturally many times (~8% of our DNA). These are all broken virus DNA now, in humans. Many of these are recognizable. Others are just viral fossils of extinct viruses. Some have accumulated so much damage that they are hard to recognize. Pigs, though, have several live viruses in their DNA. Something they had to change in pigs intended to be used as organ donors.
    DNA mutates and the genes break. If it was needed, then the people will be less fit and have less reproductive success on average, and the undamaged gene will remain the predominate form. When a gene is deleterious like the live virus, and it breaks, those with the broken form will on average have greater reproductive success. But broken genes are not removed, and accumulate. And weird additions that do nothing are often added. But they mostly come from these 3 sources: repair errors, duplication errors, and viruses.
    There would be a lot more accumulations, except that only errors in the gametes are inherited.

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  22. This looks very dangerous. 
    We have seen over the last year and a half the use of lipid nanoparticles to insert spike protein mRNA into human cells, and the extraordinary perversion of usual review mechanisms get "EUA" for experimenting on the worldwide population with a potent chemical brew after a few months research! And promoting child and pregnant women to enter the experiment!
    Lab rats can live a few months, our daughter's pet rats lasted about a year and a half, except the first one which died of a respiratory infection shortly out of the pet shop. If you compare the rat's lifespan to the human's it should take decades to confirm safety. "Efficacy" is easy if you get to define it.
    Compare the FAA Boeing 737 Max8 approval to recent "vaccine" authorization, and understand the process of "regulatory capture."
    I am a fan of elegant chemistry, but have seen enough simple drugs go wrong to have a certain scepticism. We have a High School nearby named after Frances Kelsey, who blocked US approval of thalidomide in the 1950's.

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  23. Looks like self-reply not good? Any way, I was saying that the foods eaten, etc, are influenced by the psychology, directly invoking the external problems you mention. It all mixes together.

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  24. Dan, according to the Medical Medium trauma indeed can bring a collapse of the immune system, but giving the body the optimal conditions as he subscribes to will prevent and heal the body and the soul from such a state. It goes both ways here I guess.

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  25. Little? They better be smarter than dillos, or they will suffer the same fate. Q: How many (insert your fav) does it take to eat an armadillo? A: Three. One to eat the dillo, and two to watch for cars.

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  26. "Our body, almost without exception is well designed to heal us and keep us in goodhealth, for a substantially longer time that we currently live, if we keep it at the right environment." This is true also of the internal enviro, esp chronic Repression of childhood and birth trauma. Indeed, much of the bad food and habits we kill ourselves with would be eliminated automatically if we were not using them to repress Pain in some way.

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  27. Come to think of it, the 80s Physics of Attractors aka Self Organized Dissipative Structures aka Strange Attractors stated that DNA *must* be regulated in some way by a feedback loop, probably RNA, to be stable. The junk DNA was opined to perhaps have a role. In an evolved system, it is hard to rule out hidden functionality.

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  28. I see all sorts of cases for emergency treatment. It may even be declared a right to donate one's body while alive for a long shot chance to cure or at least medical research results. Now, we need to slow down when people start laying dino eggs.

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  29. We need to get over the idea that we need to redesign our genes to fight disease. Our body, almost without exception is well designed to heal us and keep us in good health, for a substantially longer time that we currently live, if we keep it at the right environment. This is according to the Medical Medium, the greatest healer that ever lived, a man whose medical knowledge without exaggeration is more fundamental than any other form of medical of medical knowledge who receives new medical information like any other bringers of great knowledge, scientist or not, directly from the source. The nice thing about it is that his information not only helped a multitude of people self heal, but helped the medical sciences in tracing and verifying his conviction as he is re conveying concepts that science is all but too familiar with.

    The secret to achieving a state where the body can heal itself according to the Medical Medium is achieved by keeping our body clear of viruses, the root cause of all major disease that science still knows very little about, and toxins, especially heavy metals that help feed viruses and short wire the nervous system. This is true especially for the liver, that its healthy functioning regulates pathogens in the body as well as the digestion system, clean blood flow and many other functions. (Continued in my post below)

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  30. I start with a specific interest in epigenetics, but no broad knowledge of this. The recent ~20 year message has been that the junk contains some sort of pattern that the epigenetic mechanism uses to decide what to do with the *real* DNA, how to regulate it. One detracting aspect of the junk is that it is repetitive, so probably even more useless. So, look what I just found!

    https://news.wsu.edu/2021/07/23/research-identifies-potential-role-junk-dna-sequence-aging-cancer/

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  31. My understanding is that some "junk" DNA may have ancillary functions which are not well understood, but the vast majority of it is in fact without any function (except for maintaining the structure of the DNA strand).

    That is not to say, of course, that I think that going ahead with gene editing using a tool such as CRISPR is a good idea in practise. My understanding is that it is still too clumsy a hammer for the fine kind of work necessary.

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  32. I'm not the expert, but I've read that these will also edit the *junk* DNA if it happens to match. This is OK because we don't understand the junk DNA function. At least, that seems to be the logic.

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  33. I was thinking the same thing. The potential benefits to mankind are enormous. But even one minor mistake done accidentally could wreak great havoc and pain.

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