Science: CRISPR in the field of two "master" cooperation, deep analysis of single base editor off-target mechanism.

In 2012, two pioneers in gene editing, Jennifer A. Doudna and Emmanulle Charpentier, collaborated to publish a landmark paper in the history of gene editing that revolutionized the world's CRISPR-Cas9 system.
8 years, this "gene magic shear" has been widely used in agriculture, drug development and medicine at an alarming rate, and has spawned a series of gene editing tools that have become the genome editor of choice for rewriting the natural genetic code.
however, such techniques often fail to efficiently replace DNA-specific bases while ensuring that DNA double strands do not break, and the off-target effect hinders further application of the technology in the life sciences.
Recently, two "master" in gene editing, David Liu and Jennifer A. Doudna, worked together to unveil for the first time in Science the 3D structure of one of the "most promising" base editors, providing a roadmap for adjusting base editors to make them more flexible and controllable in their applications.
The new base editor "work efficiency" increases 1170 times adenine (A), bird'sine (G), cytosine (C) and thymus (T) are the basic components of DNA, and the double helix structure of DNA is constructed in the form of A-T and C-G pairings.
2016, David Liu developed the first mono-base editor to target and bind DNA without cutting DNA, replacing C-G base pairs with T-A base pairs.
, the base editor is further optimized to convert the A-T base pair back to the G-C base pair.
in human genetic diseases, the technology is expected to correct about 60 percent (more than 15,000) of disease-causing mono-base variants.
in the absence of deaminoenzymes in DNA, E. coli tRNA adenosine deaminase (TadA) was able to fuse with the Cas9 protein and evolve into ABE7.10, catalyzing the target deamination of deoxygenated adenosine.
on this basis, the ABE7.10 variant can be further improved to a shorter version of miniABEmax and ABE8e, the latest version of the Adenine Base Editor (ABE), which encodes a single TadA domain (TadA-8e) and is widely compatible with the Eight TestEd Cas Effector.
whether ABE7.10 or miniABEmax, the early adenine base editor (ABE) was inefficient.
, however, the latest ABE8e is surprisingly fast, with DNA deamino-based rates 590 times higher than ABE7.10 and miniABEmax, respectively, and 1,170 times higher.
Active deaminase protein is the "culprit" for off-targeting Although ABE8e is able to complete nearly 100% of the expected underlying edits in 15 minutes, this also means that ABE8e may be easier to edit for unrelated DNA fragments, resulting in off-target effects.
solve this problem, the researchers used cryo-EM imaging to analyze the 3D structure of ABE8e's binding DNA at a resolution of 3.2 E.
, the target adenine is replaced by a simulation designed to capture catalytic structures.
Researchers on the Cryo-EM structure when ABE8e captured DNA observed that ABE8e was more efficient because of mutations in the deaminase protein at two points compared to previous versions of the base editor, which allowed the protein to grab DNA more closely and replace G more effectively with A.
"This study shows us that this base editor actually operates as two separate modules: the Cas9 module provides specificity on the one hand, and a catalytic module that provides activity on the other," said Audrone Lapinaite, an assistant professor at Arizona State University and co-author of the report.
this provides us with a way to think about Cas9 fusion proteins, so that we know which region of Cas9 is better suited to fuse other proteins.
" activity tests show that ABE8e is prone to more off-target editing because the deaminase proteins that fuse with Cas9 are always active.
will continue to bind and release hundreds of DNA fragments before dCas9 finds its target.
, which is always active, is like an out-of-control cannon, and the dCas9 is perfectly matched to the target and "fires" at the base.
Dr Gavin Knott, a postdoctoral researcher at the University of California, Berkeley, said: "Now we can not only understand when gene editors are available and when they are not, but we can also design the next generation of basic editors to make them better and more clinical."
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