A CRISPR-free DNA editing system has been invented. It is claimed it can do effective base editing in the nucleus, mitochondria, and chloroplast genomes of plant and human cells.
The alternative system is called CyDENT and it's based on a preceding technology TALE. "TALE" stands for "transcription-activator-like effector". Before explaining what CyDENT is about, it might first be worthwhile to explain a bit about TALE. TALE proteins, which were discovered in bacteria (Xanthomonas bacteria, if you care to know), have the special property that they can be reconfigured to match any DNA sequence. To make these proteins work as gene editors, they are fused to something called a deaminase. A deaminase, in turn, is a fancy word that means a protein that can convert one DNA base to another. For example there are deaminases that convert C to T.
The shortcoming of this system is that the deaminases are themselves double-stranded pairs of DNA. The bases that they edit likewise have to be double-stranded. The new CyDENT base editing system fuses TALES with a single-strand-specific cytidine deaminase that has been found called FokI nickase.
There are some additional components: an exonuclease and a uracil glycosylase inhibitor. Exonucleases pop nucleotides off the end of a DNA strand one at a time. They are called "exo" because there's such a thing as endonucleases, which operate in the middle of a DNA strand. There are different exonucleases depending on which end of the DNA strand you want to pop, and there are exonucleases for RNA, too.
I have no idea what the uracil glycosylase inhibitor is for. A uracil glycosylase inhibitor is a protein that inhibits uracil DNA glycosylase, which is an enzyme that removes uracil from DNA. It's considered a "repair" enzyme. RNA uses uracil while DNA uses thymine. So if there's any uracil in DNA, it shouldn't be there.
Ok, at this point we turn our attention back to the main actor, the FokI nickase. It's called a "nickase" because it "nicks" DNA strands. Great name, eh? In case you're wondering, no, the "Fokl" part of the name doesn't come from the great Dr. Fokl, it comes from the name of the bacteria where it was discovered, Flavobacterium okeanokoites.
So the idea is that the TALE proteins find the target strand of DNA, the Fokl nickase "nicks" the DNA strand, exposing the desired region, the exonuclease pops nucleotides off one strand, leaving a short single stranded DNA segment. The DNA deaminase steps in here and modifies the DNA in the single-stranded segment.
If you're wondering what the point of all this is, it allows editing of single-stranded DNA in the absense of a bunch of stuff CRISPR requires, in particular the Cas9-guided R-loop structure and double-stranded DNA deaminases mentioned in the article. The R-loop structure is a complex 3-dimensional structure that forms when the Cas9 protein that the CRISPR system relies on finds its DNA target. Believe it on not, this structure involves the RNA strand that is the "guide" for the CRISPR editing process to form a "hybrid" double helix with one of the DNA strands -- the the "R-loop" actually consists of an RNA-DNA helix and a single-stranded DNA loop that is intertwined with the RNA-DNA helix.
Apparently it is really crucial for the system to be RNA-free, as that is the key to enabling it to work in the nucleus and in "organellar genomes" such as mitochondria and plant chloroplasts.
This might be a good time to tell you what CyDENT stands for, now that you'll understand all the terms in it. It stands for "cytosine deamination by nicking and editing technology".
