Ever since the start of the CRISPR craze 5 years ago, scientists have raced to invent ever-more-versatile, or efficient, variations of this powerful tool, which vastly simplifies the editing of DNA (e.g. instead of requiring 4-6 months to create a knockout mouse linem CRISPR/Cas9 methodology helps to do that task in just a few weeks). Several recently published studies [summarized in 2-page editorial, attached] have zeroed in on a more subtle approach to modifying genetic material –– which is called base-editing. One study extends a strategy for editing DNA, whereas the other study breaks new ground by base-editing DNA’s molecular cousin, RNA. Both methods open new avenues for genetic research and perhaps even curing certain genetic diseases.
CRISPR, adapted from a primitive bacterial immune system, functions first by cutting double-stranded DNA at a target site in a genome. On the other hand, base-editing does not cut the double-helix, but instead uses enzymes to precisely rearrange some of the atoms in one of the four bases that make up DNA or RNA –– converting the base into a different one without altering the bases around it; this greatly increases the options for altering genetic material. CRISPR has difficulty correcting these so-called point mutations efficiently and cleanly, thus, base editing could provide a more effective approach. Conventional CRISPR uses a guide RNA (gRNA) coupled with an enzyme known as a nuclease (e.g. most commonly Cas9) that together attach to a specific stretch of DNA bases. The nuclease then snips the double-helix. A cellular repair mechanism attempts to rejoin the cut DNA ends, but occasionally inserts or deletes bases, which alters the DNA code into gibberish and this can knock out a targeted gene
To fix a point-mutation, a CRISPR-Cas9 system must also introduce a strand of “donor” DNA that has the correct base and then rely on a second cellular mechanism called homology-directed repair (HDR). However, HDR works poorly unless cells are dividing. Base-editing systems work efficiently on non-dividing cells (e,g, brain or muscle cells). For base-editing, researchers tethered an enzyme, APOBEC1, which triggers a series of chemical reactions that ultimately change cytosine to thymine (C to T). DNA’s base-pairing “rules” specify that a T on one DNA-strand must pair with an A on the opposite strand. Then dCas9 has now been further modified to nick the unedited strand, which stimulates the cell’s DNA-repair mechanism into converting the G (that originally paired with C) into an A that pairs with the new T. Because there is no known enzyme that can convert A to G in DNA, researchers have developed one from TadA, a bacterial enzyme –– that converts A to a base called inosine, or I. Either a cellular repair mechanism or the process of the DNA copying itself then changes the I to a G. In DNA, that’s where the changes stop. In RNA, the I-containing RNA simply performs as if it had a G in that spot.
It could be several years before base-editing therapies enter clinical trials — and even longer until it’s clear whether this strategy offers advantages over existing gene therapies. So, currently, we don’t know yet whether or not base-editing is going to be a better way to approach human genetic therapy. 🙂
Science 27 Oct 2o17; 358: 432–433