Science heavy! The CRISPR field two“gurus”cooperation in depth analysis of single base editor…

Posted by btwatson on August 4, 2020 8:31 pm
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in 2012, the gene editing field of the two pioneer Jennifer A. Doudna and Emmanulle Charpentier cooperation published gene editing in the history of the milestone papers in the world brought a revolutionary CRISPR-Cas9 system. 8 years, this“gene Magic cut”at an alarming rate in the agriculture, pharmaceutical development, and medical and other fields are widely used, and derived from a series of gene editing tools, become a human rewriting the natural genetic code of choice for genome editor.

however, such techniques often cannot efficiently replace the DNA of a specific base at the same time ensure that DNA double-strand break, in addition, off-target effects also hinder the technology in the field of Life Sciences further applications.

recently, the gene editing field for two-bit“gurus”, David Liu, and Jennifer A. Doudna cooperation in “Science” for the first time opened a“most promising”of the base editor of the 3D structure, to adjust the base editor, so that in the application process more flexible and controllable to provide a road map.

DOI: 10.1126 / science. abb1390

a new version of Base Editor“work efficiency”to enhance 1170 times

adenine A, guanine G, and cytosine C and thymine（T）is DNA the basic structural unit, according to A-T, C-G pairing in the form to build up the DNA double helix structure.

in 2016, David Liu developed the first single-base editor, it is possible without cutting the case of DNA, the targeting and binding to DNA, the C-G base pairs replaced with T-A base pairs. A year later, the base editor was further optimized, the A-T base pair is converted back to G-C base pairs. In human genetic diseases, the technology is expected to correct about 60% of the more than 15,000 species of pathogenic single-nucleotide variation.

in the DNA is not deaminated the case of enzymes, the E. coli tRNA adenosine deaminase（TadA to be able to with the Cas9 protein fusion evolved into ABE7. 10, the catalytic deoxy adenosine targeting the release of ammonia. On this basis, ABE7. 10 variants can be further improved to become a truncated version of the miniABEmax and ABE8e, which is the latest version of the adenine bases editor, ABE, and capable of encoding a single TadA domains TadA-8e, and with eight test Cas effector widely compatible.

whether it is ABE7. 10 or miniABEmax, the early adenine nucleotide editor ABE efficiency is very low. However, the latest ABE8e’re fast to surprisingly, the DNA deamination rate than ABE7. 10 and miniABEmax, respectively, higher than 590 times and 1170 times.

active deaminase protein is caused by off-target the“culprit”

although ABE8e can be done in 15 minutes almost 100% of the expected based editing, but it also means that ABE8e may be easier for independent DNA fragments to be edited, resulting in off-target effects.

in order to solve this problem, the researchers use of the frozen electron microscopy（Cryo-EM）imaging techniques to 3. 2 Å resolution analysis of the ABE8e DNA binding when the 3D structure. Wherein, the target adenine is designed to capture the catalytic conformation of the mimetic replaced.

ABE8e capture DNA when the Cryo-EM structure

researchers observed that ABE8e the reason why having a high operating efficiency, because with the previous version of the base editor compared to the deaminase protein in the two point position on the presence of the mutation, which makes��White matter can be more tightly grab the DNA, and more effectively G is replaced A.

the report of the co-first author, Arizona State University assistant Professor Audrone Lapinaite, said:“this 3D structure allows us to understand that the base editor is actually two independent modules running: on the one hand Cas9 module provides specific, on the other hand the presence of a catalytic module providing the activity. This provides us with a thinking Cas9 fusion protein method, so that we know the Cas9 which area is more suitable for Fusion with other proteins.”

activity testing showed that ABE8e the reason is easy to produce more off-target editing, is because with the Cas9 fusion of the deaminase protein is always in the active state. In the dCas9 to find the target before, will continue to bind and the release of hundreds of DNA fragments. Always in the active state deaminase like a runaway cannons, ranging dCas9 completely matched to the target it is already on the base“fire”was.

University of California at Berkeley post-doctoral researcher Gavin Knott, Dr. said:“now, not only can we learn about the gene editor when you can use, and when not to use, but also can design the next generation based editor to make them better, more suitable for clinical use.”

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End

reference:

[1] DNA capture by a CRISPR-Cas9–guided adenine base editor.

[2] Cryo-EM Captures the CRISPR-Cas9 Base Editor in Action.

[3]the United States the Broad Institute David Liu: adenine nucleotide editor ABE）

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Published at Mon, 04 Aug 2020 11:24:45 +0000

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