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Tightly Controllable Cas9 Genome Editor Could be Useful for Gene Drive Regulation

NEW YORK (GenomeWeb) – Researchers from the University of Bath and Cardiff University in the UK have developed a way to tightly control CRISPR-Cas9-based genome editing using a cheap derivative of lysine. They noted that such a system could have broad applications, and that it is a first step towards purposed, spatiotemporal regulation of gene drives over large geographical ranges.

Tight control of Cas9-based genome editing would minimize unintended and potentially harmful genome cleavage due to leaky nuclease activity, the researchers said in their study. Such regulation could certainly restrict genome editing to given anatomical sites in clinical somatic cell gene therapy, and it could also do the same thing in gene drives, which have the potential to eliminate destructive invasive species and agricultural pests. However, the authors noted in their study in Scientific Reports this week, gene drives require "remarkable control."

Previous methods to control Cas9 activity have involved modulating light or temperature, but these factors are difficult to regulate, the researchers noted. Certain small molecule effectors have also been used, including rapamycin, trimethoprim and (Z)-4-hydroxytamoxifen. But rapamycin and trimethoprim are antibiotics and could drive antimicrobial resistance, while (Z)-4-hydroxytamoxifen — which is the major active metabolite of tamoxifen — is an endocrine disruptor and aquatic toxin. "The cost of small molecule regulators in financial and environmental terms is also likely to be prohibitive on large scales," the authors wrote.

To address these issues, the investigators instead turned to a cheap amino acid — the lysine derivative H-Lys(Boc)-OH (BOC) —to control Cas9. "BOC can be incorporated into proteins of interest by genetic code expansion, suggesting that a novel tier of regulating Cas9 might be achieved by generating a functional Cas9 variant whose expression (translation) and activity depended on the presence (incorporation during translation) of BOC," they wrote. "We therefore set out to evaluate the control of Cas9 by genetic code expansion in mammalian heritable genome alteration as a first step to the stringent control of gene drives and other applications."

To start, the team characterized genetic code expansion to control the expression of eGFP in transgenic mouse embryos, and found that RNA injection resulted in efficient BOC-inducible expression of green fluorescence via eGFP in oocytes.

They then wanted to evaluate whether genetic code expansion could be used to control the RNA-guided nuclease activity of Cas9 by adding BOC. Since Cas9 residues can be substituted with a bulky Lys derivative without abolishing endonuclease activity, the researchers tested Cas9 in which the lysines K510 or K742 were replaced with BOC residues. They evaluated this system in HEK293 cells transfected with constructs encoding eGFP gRNA, and found that transfectants exposed to BOC expressed Cas9 mutants and a marked reduction of eGFP protein levels, suggesting BOC-induced Cas9K510B eGFP gene targeting activity.

Through a series of further experiments in mice, the researchers also tracked the effect of BOC-Cas9 editing system's efficiency, and found that blastocyst development rates were similar in both groups: about 63.3 percent of injected oocytes developed in the absence of BOC compared to about 64.4 percent of oocytes with it. When BOC was included, 7.4 percent of the resulting blastocysts lacked fluorescence and contained edited eGFP transcripts, whereas all of the blastocysts fluoresced brightly when it was omitted. "Given that the control editing rate was 21.2 percent, BOC-dependent editing by Cas9K510B transgene-expressing females had therefore occurred at about 34.9 percent of the control rate (7.4 percent for Cas9K510B vs. 21.2 percent for the control)," the authors wrote.

They also noted that the BOC system was compatible with efficient full-term development of the mice, even when active at an early and very sensitive phase of embryogenesis. This shows that there is likely no extensive off-target editing in the system. "In contrast to the higher background activities or restricted applicability of many switchable systems, this work accordingly demonstrates low or zero Cas9BOC activity in the absence of BOC; without BOC, Cas9BOC activity was undetectable under the conditions of these experiments, suggesting that the system was subject to little, if any, leakiness," the authors wrote.

They concluded that BOC-inducible Cas9 in gametes or embryos near the time of fertilization could mediate tight gene drive regulation. And because BOC is a cheap amino acid, it could potentially be used in the environment to control gene drive expression. Further, the team generally used low concentrations of BOC, which are expected to exert fewer co-lateral effects and reduce costs.

The researchers also noted that BOC-regulated Cas9 could be used for human biomedical applications. "They would, for example, enhance spatiotemporal control of targeted viral integration and/or activity in somatic cell therapies (by restricting BOC to a given anatomical site and time) and mitigate against neutralization by pre-existing anti-Cas9 antibodies," they wrote. "This control principle holds for proteins other than Cas9."

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