Retron Library Recombineering

June 21, 2021 10:49 PM

Scientists Have Created A New Gene-Editing Tool That Could Outperform CRISPR - "It is faster and simpler than CRISPR, enabling millions of genetic experiments to be performed simultaneously."

This tool, described in a recent paper in PNAS, employs retrons, which are bacterial DNA segments that undergo reverse transcription to generate single-stranded DNA fragments (ssDNA). RLR produces up to millions of mutations concurrently in bacterial cells and "barcodes" mutant cells, enabling the whole pool to be screened at once. This way large quantities of data can be quickly produced and analyzed.

But why is this important? Well, because it overcomes the major limitations of CRISPR-Cas9, a groundbreaking technology that can be used to edit genes. Overall, it is difficult for scientists to deliver CRISPR-Cas9 materials in large numbers, and it can sometimes be toxic to cells since the Cas9 enzyme, the molecular "scissors" that cut strands of DNA, often cuts unintended sites...

While CRISPR-Cas9 cuts DNA to insert the mutant sequence into its genome, retrons can insert the mutant DNA strand into a replicating cell, where it would be introduced into the DNA of the daughter cells. Moreover, since sequences of retrons can be used as "barcodes," this enables scientists to track individuals... All in all, the advancements are exciting but may be a little premature. RLR is yet to work in mammalian cells.

also btw...

• Scientists Develop New Gene Editor to Correct Disease-Causing Mutations - "A team invented a CRISPR-based gene editor that precisely changes the defective C within the genome to the desired G. This C-to-G base editor (CGBE) invention opens up treatment options for approximately 40 percent of the single-base substitutions that are associated with human diseases such as the aforementioned cystic fibrosis, cardiovascular diseases, musculoskeletal diseases, and neurological disorders."
• Artificial Proteins Never Seen in the Natural World Are Becoming New COVID Vaccines and Medicines - "Researchers have begun to crack the code of protein structure, allowing them to remake, well, everything."
• First cells reprogrammed to make synthetic polymers—and virus-resistant drug manufacturers - "Scientists have developed the first cells that can construct artificial polymers from building blocks that are not found in nature, by following instructions the researchers encoded in their genes."
• Scientists Used CRISPR to Engineer a New 'Superbug' That's Invincible to All Viruses - "Can we reprogram existing life at will? ... The team used CRISPR to replace over 18,000 codons with synthetic amino acids that don't exist anywhere in the natural world. The result is a bacteria that's virtually resistant to all viral infections—because it lacks the normal protein 'door handles' that viruses need to infect the cell. But that's just the beginning of engineering life's superpowers."
• First Major Discoveries Reported From Project to Read Complete Genetic Sequences of All 70,000 Vertebrate Species - "'We will get a spectacular picture of how nature actually filled out all the ecosystems with this unbelievably diverse array of animals'... The VGP model is even inspiring other large sequencing efforts, including the Earth Biogenome Project, which aims to decode the genomes of all eukaryotic species within 10 years."
• Plankton have a genome like no other
• Study uses plankton genomes as global biosensors of ocean ecosystem stress
• Bioengineers Develop Algorithm to Compare Cells Across Species – With Striking Results - "Researchers created an algorithm to identify similar cell types from species – including fish, mice, flatworms and sponges – that have diverged for hundreds of millions of years, which could help fill in gaps in our understanding of evolution. Cells are the building blocks of life, present in every living organism. But how similar do you think your cells are to a mouse? A fish? A worm?"
• 24,000-year-old organisms found frozen in Siberia can still reproduce
• How 'Great' Was the Great Oxygenation Event? Enzyme 'Family Tree' Reveals When Organisms First Started Using Oxygen
• Biologists Construct a 'Periodic Table' for Cell Nuclei – And Discover Something Strange, Baffling and Unexpected - "A team of biologists studying the tree of life has unveiled a new classification system for cell nuclei, and discovered a method for transmuting one type of cell nucleus into another."

posted by kliuless (9 comments total)

the Cas9 enzyme, the molecular "scissors" that cut strands of DNA, often cuts unintended sites

These are so-called "off-target" effects, where mismatches between the target sequence and enzyme result in cuts made where you don't want them, and so mutations are added where you don't want them. Depending on the application, unwanted and uncontrolled mutations can have very problematic consequences. TALEN-based editing has been around longer, despite getting less media coverage. It has fewer of these off-target effects, but it is a more difficult platform to set up and can also have decreasing efficiency, depending on where target sites are located along a gene of interest, though as a platform it tends to be more efficient than CRISPR, especially in more compressed regions of the genome.
posted by They sucked his brains out! at 2:01 AM on June 22, 2021 [5 favorites]

Outperform in a very limited setting. If you're already worried about off-target effects you probably don't want to introduce millions of mutations at once, even if each individual cell only gets one or two mutations.

If you're trying to evolve bacterial/cell line populations though, sure. This could be really interesting.
posted by Orange Pamplemousse at 4:14 AM on June 22, 2021

If you want to have a twitter feed to learn about gene editing and base editing by one of the experts in base-editing (individual edits, David Liu of Harvard is a good place to start.

The potential of base editing and genome editing can't be overstated. Here are two real, validated discoveries that have an actual path to the clinic; if they work they will save tens of thousands and tens of millions of lives respectively.

Treating and potentially curing sickle cell anemia.


Permanently knocking one of the genes responsible for high cholesterol in primates
posted by lalochezia at 5:00 AM on June 22, 2021 [5 favorites]

It wasn't very clear to me from the article why making a bacteria that is resistant to all viruses would be a good thing?
posted by aniola at 9:57 AM on June 22, 2021 [1 favorite]

Because they can, and because it communicates their opinion of the possibilities of the technique. I don't know that anyone is proposing such a thing. [Cue Ian Malcolm]
posted by eustatic at 10:28 AM on June 22, 2021

Thrilling. Terrifying.
posted by Corvid at 6:42 PM on June 22, 2021

Yeah, the not so perma frost. Life is change, as long as change is survivable. Interesting how the potential for major change sits in labs and the background singers are all about heroic effects, and the money swirls about their ankles, to fund every sort of actor with enough skills. Idealists come in every persuasion. The battles are huge, complicated like oil vs the sky, the clean fresh air we need vs every last thing. Then the old saying attributed to Mark Twain, about whiskey, "Too much is just enough." Then the we and them thinking, Then geo something or othering the sky to make up for what we are going to keep on doing. Then you will have to purchase a successful child's DNA, so your family can maintain their status? I am generally unnerved at our atrocious ways. Glad I am older. I am fully depreciated.
posted by Oyéah at 9:07 PM on June 22, 2021

ugh the Fermi paradox is starting to make a lot more sense.
posted by wibari at 9:48 PM on June 22, 2021

Mind blowing. Thank you so much kluiless for the fantastic posts. (At least somebody is alive in here.)
posted by blue shadows at 11:17 PM on June 22, 2021