• LW - Multiplex Gene Editing: Where Are We Now? by sarahconstantin

  • Jul 16 2024
  • Duración: 14 m
  • Podcast

LW - Multiplex Gene Editing: Where Are We Now? by sarahconstantin

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  • Welcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: Multiplex Gene Editing: Where Are We Now?, published by sarahconstantin on July 16, 2024 on LessWrong. We're starting to get working gene therapies for single-mutation genetic disorders, and genetically modified cell therapies for attacking cancer. Some of them use CRISPR-based gene editing, a new technology (that earned Jennifer Doudna and Emmanuelle Charpentier the 2020 Nobel Prize) to "cut" and "paste" a cell's DNA. But so far, the FDA-approved therapies can only edit one gene at a time. What if we want to edit more genes? Why is that hard, and how close are we to getting there? How CRISPR Works CRISPR is based on a DNA-cutting enzyme (the Cas9 nuclease), a synthetic guide RNA (gRNA), and another bit of RNA (tracrRNA) that's complementary to the gRNA. Researchers can design whatever guide RNA sequence they want; the gRNA will stick to the complementary part of the target DNA, the tracrRNA will complex with it, and the nuclease will make a cut there. So, that's the "cut" part - the "paste" comes from a template DNA sequence, again of the researchers' choice, which is included along with the CRISPR components. Usually all these sequences of nucleic acids are packaged in a circular plasmid, which is transfected into cells with nanoparticles or (non-disease-causing) viruses. So, why can't you make a CRISPR plasmid with arbitrary many genes to edit? There are a couple reasons: 1. Plasmids can't be too big or they won't fit inside the virus or the lipid nanoparticle. Lipid nanoparticles have about a 20,000 base-pair limit; adeno-associated viruses (AAV), the most common type of virus used in gene delivery, has a smaller payload, more like 4700 base pairs. 1. This places a very strict restriction on how many complete gene sequences that can be inserted - some genes are millions of base pairs long, and the average gene is thousands! 2. but if you're just making a very short edit to each gene, like a point mutation, or if you're deleting or inactivating the gene, payload limits aren't much of a factor. 2. DNA damage is bad for cells in high doses, particularly when it involves double-strand breaks. This also places limits on how many simultaneous edits you can do. 3. A guide RNA won't necessarily only bind to a single desired spot on the whole genome; it can also bind elsewhere, producing so-called "off-target" edits. If each guide RNA produces x off-target edits, then naively you'd expect 10 guide RNAs to produce 10x off-target edits…and at some point that'll reach an unacceptable risk of side effects from randomly screwing up the genome. 4. An edit won't necessarily work every time, on every strand of DNA in every cell. (The rate of successful edits is known as the efficiency). The more edits you try to make, the lower the efficiency will be for getting all edits simultaneously; if each edit is 50% efficient, then two edits will be 25% efficient or (more likely) even less. None of these issues make it fundamentally impossible to edit multiple genes with CRISPR and associated methods, but they do mean that the more (and bigger) edits you try to make, the greater the chance of failure or unacceptable side effects. How Base and Prime Editors Work Base editors are an alternative to CRISPR that don't involve any DNA cutting; instead, they use a CRISPR-style guide RNA to bind to a target sequence, and then convert a single base pair chemically - they turn a C/G base pair to an A/T, or vice versa. Without any double-strand breaks, base editors are less toxic to cells and less prone to off-target effects. The downside is that you can only use base editors to make single-point mutations; they're no good for large insertions or deletions. Prime editors, similarly, don't introduce double-strand breaks; instead, they include an enzyme ("nickase") that produces a single-strand "nick"...
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