A Crispr-Cas9 System Designed to Introduce Point Mutations into the Human ACE2 Gene to Weaken the Interaction of the ACE2 Receptor with the SARS-CoV-2 S Protein
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How to cite: Tanaka, P.; Santos, J.; Oliveira, E.; Miglioli, N.; Assis, A.; Monteleone-Cassiano, A.; Ribeiro, V.; Duarte, M.; Machado, M.; Mascarenhas, R.; Souza, A.; Brito, L.; Oliveira, L.; Donadi, E.; Passos, G. A Crispr-Cas9 System Designed to Introduce Point Mutations into the Human ACE2 Gene to Weaken the Interaction of the ACE2 Receptor with the SARS-CoV-2 S Protein . Preprints 2020, 2020050134. https://doi.org/10.20944/preprints202005.0134.v1 Tanaka, P.; Santos, J.; Oliveira, E.; Miglioli, N.; Assis, A.; Monteleone-Cassiano, A.; Ribeiro, V.; Duarte, M.; Machado, M.; Mascarenhas, R.; Souza, A.; Brito, L.; Oliveira, L.; Donadi, E.; Passos, G. A Crispr-Cas9 System Designed to Introduce Point Mutations into the Human ACE2 Gene to Weaken the Interaction of the ACE2 Receptor with the SARS-CoV-2 S Protein . Preprints 2020, 2020050134. https://doi.org/10.20944/preprints202005.0134.v1
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Unfortunately, this methodology is entirely insufficient to substantiate the claims proposed by the authors. In order to actually predict with some degree of confidence that the back-bone conformation of ACE2 will not change significantly upon the introduction of the engineered mutations, the authors must conduct homology modeling of the whole protein followed by at least traditional molecular dynamics simulations and RMSD/RMSF reporting. Ideally, the authors would generate multiple homology models capturing the different possible rotamers of each of the mutated side-chain, then evaluate each of them through a metadynamics pipeline in comparison to the wild-type structure.
In addition, protein/protein docking of ACE2 to the binding domain of the S envelope protein followed by umbrella sampling to estimate potential of mean force would add significant strength to the claims presented here, if the author's hypothesis is correct it would be trivial to capture the shifts in binding energy between wild-type ACE2 and their proposed mutants.
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We will still have to test this system in vitro to see if the proposed changes interfere with the adhesion of the SARS-CoV-2 virus.
Thanks for the constructive comments.
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We will still have to test this system in vitro to see if the proposed changes interfere with the adhesion of the SARS-CoV-2 virus.
Thanks for the constructive comments.
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Unfortunately, it is not clear the advantages of using this tool over vaccines and soluble ACE2 in the context of SARSCOV2 infection.
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In this manuscript, we decided not to speculate on possible advantages of the proposed Crispr-Cas9 model over an anti-COVID-19 vaccine, which has not been launched yet. An effective vaccine will always be welcome, and it is the gold standard for immunological prevention against infectious diseases. The model we propose has a different mechanism than a vaccine. We suggest gene editing so that the ACE2 protein decreases its interaction with SARS-CoV-2 without losing its regular physiological activity, the conversion of angiotensin 2. As discussed by Ciaglia (2020) (DOI: 10.3389/fped.2020.00206), "circulating ACE2 enzyme offers protection against influenza (H7N9) virus acute lung injury (Yang et al. 2014) (DOI: 10.1038/srep07027). Note that this was done to the influenza A (H7N9) virus, so it is not yet possible to speculate whether our gene editing model would be better or worse than treatment with soluble ACE2 in cases of COVID-19.
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How the authors think that using CRISPR to edit the DNA from everybody would be possible? I mean, this still very expensive and we have 7 billion people in the world. How much it would cost and how much time would take to edit everybody, considering that just a few places are able to do it? What are the consequences of having a editing in the off-targets listed?
Commenter:
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This type of questioning is valid for all strategies based on Crispr-Cas9 for the treatment of human diseases.
We have no answer to all questions. In the preprint that we have published, we have tried to show a possibility to edit a specific part of the ACE2 gene that encodes the Sars-CoV-2 receptor, weakening the link with the virus. We have already provided the reagents, host cell line, and virus line to test this idea in laboratory practice.
We still do not know if the bioinformatics predictions of this work will be confirmed in practice. We hope so, but the experiments are at an early stage. Therefore, we have not yet speculated its use in human populations. This is not an easy subject to be treated, as a new type of treatment has to be tested before in phase I, phase II, and phase III. Another important point to be considered, as the questioner himself pointed out, is the possible side effects arising from changes in the off-targets. We will have to work on that too. As you can see, we demonstrate a concept that encourages various aspects of scientific research with the possibility of application. Thanks again for the question.
Geraldo A. Passos, PhD