Approach improves avoidance of ‘viewer’ changes in CRISPR-based editor processing – ScienceDaily

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Want to keep the scum out of the gene pool party? Sneak up and slam the door before they arrive.

This is the central idea of ​​a new strategy from scientists at Rice University who seek to avoid gene editing errors by adjusting specific CRISPR-based editing strategies in advance.

Rice chemical and biomolecular engineer Xue Sherry Gao and chemist Anatoly Kolomeisky and their labs combined theory and experimentation for a holistic approach to create better basic editors, molecular machines that target and repair defective DNA at a single base resolution.

Their work appears in Natural communications.

The article describes the molecular processes that base editors use to manipulate DNA strands, cutting them off if necessary and making way for the replacement code. When it works, as it increasingly does to treat genetic diseases like sickle cell anemia and certain cancers, the publisher only changes the intended nucleotide.

And when it doesn’t, it’s because changes made by viewers can cause unwanted effects.

The Rice strategy mainly seeks to eliminate the wayward edits of the viewers, the nucleotides adjacent to the target of the base editor. Gao’s lab previously introduced tools to improve the accuracy of CRISPR-based modifications of cytosine mutations up to 6,000-fold.

For the new project, she hired the Kolomeisky Lab to help create a theoretical framework to eliminate trial and error in the design of an editor library. These would better target the mutations that cause the disease while avoiding passers-by. In the process, the framework could help scientists better understand the chemical and physical processes that take place when editing the database.

“Sherry and other experimental scientists already had results that worked,” Kolomeisky said, referring to the previous article, in which the lab used its editor to convert cytosines to thymines, correcting DNA mutations while by avoiding otherwise vulnerable cytosines upstream. “But despite these amazing developments, there has been no microscopic understanding of what we need to do with these protein systems to improve editing.”

He said that Qian Wang, a former postdoctoral researcher in Kolomeisky’s lab and now an assistant professor at China University of Science and Technology (USTC), Hefei, rose to the challenge by using Gao’s cytosine experiment as a reference.

“We applied the model for this result and got a few important parameters which we then used to design which mutations and where are needed to achieve precise editing,” Kolomeisky said. “Ultimately, this symbiosis of theory and experimentation allows us to work intelligently.”

Their strategy combines molecular dynamics simulations and stochastic (aka random) models that identify the binding energies between molecules required to achieve maximum editing selectivity. Experiments in Gao’s lab validated the results.

Critically, the framework includes a way to characterize the binding affinity between deaminases – enzymes that catalyze the removal of an amino group from a molecule – and single-stranded DNA (ssDNA).

Ideally, they said, the deaminase stays on the sDNA just long enough to complete the major editing and is released before inadvertently editing a control site.

“The important thing here is that a mutation doesn’t work for different systems,” Kolomeisky said. “So for each system you have to redo this procedure, but at least what needs to be done is clear.”

“The model was very successful at reflecting what has already been done experimentally,” Gao said. “But since then we have been able to refuse viewer effects in other basic editing systems.

“Because the number of mutants could run into the thousands, it is unrealistic for experimenters alone to verify individual base editors,” she said. “Only this multidisciplinary approach will allow us to build a huge library of desktop editors, and then narrow the numbers down to the most promising candidates for further experimental checks. That’s what we’re working towards.”

Postdoctoral researcher Rice Jie Yang is co-lead author of the article with Wang. Rice’s undergraduate Jeffrey Vanegas and USTC theoretical physicist Zhicheng Zhong are co-authors of the article. Gao is Ted N. Law Assistant Professor in Chemical and Biomolecular Engineering. Kolomeisky is Professor and Chairman of the Department of Chemistry and Professor of Chemical and Biomolecular Engineering.

Double First-Class Initiative USTC research funds (YD2030002006), National Natural Science Foundation of China (32000882), National Science Foundation (NSF) (1953453, 1941106), Center for Theoretical Biological Physics (19745) funded by NSF), Welch Foundation (C-1559), National Institutes of Health (R01HL157714) and Rice Creative Ventures Fund supported the research.


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