4000 Times Fewer Gene Editing Errors Without Sacrificing Speed

Scientists at The University of Texas at Austin have redesigned a key component of a widely used CRISPR-based gene-editing tool, called Cas9, to be thousands of times less likely to target the wrong stretch of DNA while remaining just as efficient as the original version, making it potentially much safer.

Other labs have redesigned Cas9 to reduce off-target interactions, but so far, all these versions improve accuracy by sacrificing speed. SuperFi-Cas9, as this new version has been dubbed, is 4,000 times less likely to cut off-target sites but just as fast as naturally occurring Cas9. Bravo says you can think of the different lab-generated versions of Cas9 as different models of self-driving cars. Most models are really safe, but they have a top speed of 10 miles per hour.

“They’re safer than the naturally occurring Cas9, but it comes at a big cost: They’re going extremely slowly,” said Bravo. “SuperFi-Cas9 is like a self-driving car that has been engineered to be extremely safe, but it can still go at full speed.”

So far, the researchers have demonstrated the use of SuperFi-Cas9 on DNA in test tubes. They’re now collaborating with other researchers who plan to test SuperFi-Cas9 for gene editing in living cells. They’re also working to develop still safer and more active versions of Cas9.

Taylor and Johnson developed a technique called kinetics-guided structure determination that used a cryo-electron microscope in the Sauer Structural Biology Lab to take snapshots of Cas9 in action as it interacted with this mismatched DNA.

They were surprised to discover that when Cas9 encounters this type of mismatch in positions 18 through 20, instead of giving up and moving on, it has a finger-like structure that swoops in and holds on to the DNA, making it act as if it were the correct sequence. Normally, a mismatch leaves the DNA a bit floppy; this finger-like structure stabilizes it.

“It’s like if you had a chair and one of the legs was snapped off and you just duct taped it together again,” Bravo said. “It could still function as a chair, but it might be a bit wobbly. It’s a pretty dirty fix.”

Based on this insight, they redesigned the extra finger on Cas9 so that instead of stabilizing the part of the DNA containing the mismatch, the finger is instead pushed away from the DNA, which prevents Cas9 from continuing the process of cutting and editing the DNA. The result is SuperFi-Cas9, a protein that cuts the right target just as readily as naturally occurring Cas9, but is much less likely to cut the wrong target.

Nature – Structural basis for mismatch surveillance by CRISPR–Cas9

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed these suffer from substantially reduced rates of on-target DNA cleavage. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

SOURCES- University of Texas, Nature
Written by Brian Wang, Nextbifuture.com

15 thoughts on “4000 Times Fewer Gene Editing Errors Without Sacrificing Speed”

  1. Given the current events, we could have the Western Government colluding with Big Pharma, to inject people with such a technology in the guise of some "mandatory" vaccination for some puny virus.
    80% of the population would obey without hesitation.
    In the future with more than hesitation, but it is another story.

  2. The evil regime convicts you of a "crime" and then harvests your body parts. There does not seem to be an exchange policy.

  3. Have you read an account of what it took for Elizabeth Parrish the CEO who modified herself to modify herself? She had to have dozens of injections, and it requires a virus to infect the cells, so she had to get quite sick for the little bit of change she did get.
    This is not something that can be done sneakily.
    I tried to find the video, but there are just too many. I only remember her telling what the ordeal was once…so I can't say the number of injections it was, but it was a lot. The rest of the videos it is just about how great this is going to be and the results she is getting.
    And you almost certainly have to be very healthy already to endure the procedure.

    Why would some government want to secretly mess with everyone's epigenome? What is in it for them?
    You think our government wants to make our healthcare cheaper? They are actively making it more expensive, as healthcare companies and insurance companies donate to campaigns.
    And if they want to damage our health, there are much easier ways. Like delaying the ban on trans fat for over a decade past the presentation of clear evidence that it was killing 50,000 Americans a year…like they did. Or failure to do a thing about organphoshate fertilizers that are causing brain damage…or the likely hundreds of other harmful chemicals floating around.
    Even cinnamon can be harmful. There are 2 types and the cheap one used in every commercial product is the harmful one. You want ceylon cinnamon. Nothing is done.

  4. Good, I wish you're right and this is just me being paranoid and disinformed.

    But in fact it is much simpler to inject some practically undetectable DNA-changing nanotech in a vaccine than going around swapping people's hearts. We have already seen how such therapies can arrive to a big population relatively quickly and the reasons why.

    Several aspects of human behavior are epigenetic or genetics related, and this is why a massive population modification might be of interest for some governments.

    And I presume they are taking the circulatory system effects into account or this won't be of much general use.

  5. What would I do? I think ideal is genetic repair in the gonads. Then you can just have children naturally, or at least optionally. You could still go further and select an embryo. But I don't think that would be necessary. That is mostly insurance against large chromosomal defects…and more important when the mother is over 40. I think it is nonsense that you can pick an embryo with 20 more IQ points than another. I think IQ up or down is mostly due to nutrition, infection, hypoxia (during birth), toxins, and chance timely brain connections. Scientists looking for intelligence genes, using over 78,000 people, could only account for 5% of the difference in IQ scores.
    And despite the fact that they can't account for much, they still assume that the studies showing 80% genetic contribution are true.
    I think it is far more parsimonious that the test showing 80% was flawed in that they used twins separated at birth, and did not account for the fact that they shared the same intrauterine environment for 9 month as well as genetics. It is that environment, I believe, that matters by far the most. And we see this. The unborn are strongly affected by toxins and viral infections. Mom eating 5 servings of fruit daily gives 5 IQ points. Choline beyond recommended makes similar gains. And 95% don't even get the recommended. Similar stories with iodine. Even random things like eating black licorice can drop a bunch of IQ points. How many food effects are we unaware of?

  6. I had a heart murmur when I was a toddler. I grew out of it. Never thought that much about it, until I saw it in my genome. No big deal, I suppose. And when I was very young, I did not hold down food well. Why, is unclear. I suppose it could have been genetic. That could be why I am shorter than my brother. Perhaps why I went to Kindergarten a year late.
    I am probably exaggerating the effects, but you think of these things when you consider that if you had children, they too may have something you had.
    What child with myopia has not been frustrated by glasses? I am pretty myopic, have astigmatism too. Any future partner would probably see that right off the bat, as I wear glasses. But kind of a bummer that any children will likely have it.
    Anyone can probably list at least 5 things about them, that are clearly flaws, that conceivably can be fixed in a future generation…if we are not terrified.
    "More than 32 percent of the world’s population has myopia, or nearsightedness, according to a 2019 study in the Investigative Ophthalmology and Visual Science journal. The study predicts that by 2050, almost 60 percent of the global population will have some level of myopia, predominantly in developed countries."
    If we do nothing, it is conceivable that virtually everyone will be myopic in a few hundred years. Except Cambodia, perhaps, where they killed everyone with glasses.
    Since 2000, it has increased by almost 14% in the US. Should we laser everyone to the end of time, instead?

  7. No one will update their thinking about genetic surgery of embryos even if changes in the technology warrant a rethink. It often takes another generation to say: "Hey, your reasoning is rubbish, pops.", when people have been duped. We were duped out of nuclear power. We were duped out of decent infrastructure, and homes by NIMBYs, and often misguided environmentalists. We were duped out of our factories and good jobs by union organizing con-men. We have been duped out of single payer by lies about cost and economics. We were duped out of just reasonable basic government services, by lawyers, so they can "win" large awards in lawsuits against cities, counties and States. Juries thoughtlessly, award 10s or 100s of millions of dollars in cases where some government employee did something terrible. Put that egregious employee away. The idea that we should double taxes to pay for someone's incompetence, corruption, or malevolence to make the victim or the victim's family rich, and of course the lawyer rich, with his fat cut, is idiotic. We have been had. If it is "systemic", fire a bunch of people…until it is not.

    Why should people be born with genetic defects, if it can be fixed? And I don't think God will appreciate you giving him credit for something terrible that we could have fixed, and likely caused with chemicals.

    1 in 33 babies is born with a serious genetic defect: https://www.marchofdimes.org/baby/rare-birth-defects.aspx

    There are over 7,000 kinds. They add up.

  8. Nonsense. Genetic modification rarely affects more than 1 or 2% of cells…and mostly in the circulatory system or the blood. Such a modification would have limited affect, and be very detectable.
    This is like after it became possible to replace a heart with another, to go around saying there is a danger some evil regime is going to take your heart out and give you another…just because it is possible.

  9. It's great that they got run speeds up, but some are still worried about the error rates still. Kinematics is still a difficult problem for computational chemistry guys.

  10. A very important story I’d missed. This is a great example of the best of Next Big Future – especially for breakthroughs at the edge of science and engineering.

    Cas9 was a fantastically important discovery, a molecular machine evolved in bacteria as a sort of tiny anti-viral immune system. Humans then put it to use as genetic editing nano machinery, but it’s original function wasn’t for precision editing but just to identify invasive code and cut it up to neutralize it. It didn’t evolve to be ideal for what we use it for.

    ReEngineering it to significantly increase precision while keeping it’s speed is a historic step.

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