Preprint Article Version 1 This version is not peer-reviewed

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

Version 1 : Received: 6 May 2020 / Approved: 7 May 2020 / Online: 7 May 2020 (15:26:06 CEST)

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 (doi: 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 (doi: 10.20944/preprints202005.0134.v1).

Abstract

The human angiotensin-converting enzyme 2 (ACE2) has a crucial role on blood pressure control; however, ACE2 is also the primary SARS-CoV-2 (S domain) virus receptor. Inhibiting or even reducing the expression of the native ACE2 might diminish the viral entry into the cells, but may cause a failure of ACE2 biological activity, primarily in patients with comorbidities, including diabetes mellitus or hypertension. Since the ACE2 catalytic site and the SARS-Cov-2 receptor are distinct, we designed a Crispr-Cas9 model system, predicting the respective sequences for a guide RNA (gRNA) and a single-stranded oligo dideoxy nucleotide (ssODN), to introduce point mutations into the exon 1 of the human ACE2 gene, which encodes the alpha-helix, implicated on the binding of the SARS-CoV-2 envelope S protein. Protein modeling predicted that the specific substitutions of residues Phe28, Lys31, and Tyr41 for Ala at the ACE2 alpha-helix do not significantly alter ACE2 native conformation. The analysis of the impact of these mutations on ACE2 receptor function predicted a weakening of the binding of the SARS-CoV-2 protein S. An experimental genome editing of cells based on these Crispr-Cas9 elements might reduce the SARS-CoV-2 ability to enter the epithelial cell, preserving the biological activity of ACE2 enzyme.

Supplementary and Associated Material

Subject Areas

Crispr-Cas9; ACE2 gene; SARS-CoV-2; new coronavirus; COVID-19

Comments (7)

Comment 1
Received: 13 May 2020
The commenter has declared there is no conflict of interests.
Comment: The authors claim that introducing mutations to residues Phe28, Lys31, and Tyr41 does not significantly alter ACE2 native conformation. This assumption is based on molecular modeling of the helical peptide of which these residues are part of.

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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Response 1 to Comment 1
Received: 14 May 2020
Commenter: Jadson C. Santos
The commenter has declared there is no conflict of interests.
Comment: We appreciate your feedback as it is very important for the follow-up of our work. Regarding the analyzes indicated in your comments, they are part of our planning. Thank you again.
Response 2 to Comment 1
Received: 14 May 2020
Commenter: Geraldo Aleixo Passos
The commenter has declared there is no conflict of interests.
Comment: Another important aspect for this project are the in vitro experiments, to check if the edited cells became at least in part, refractory to Sars-cov-2 infection. We are planning experiments to test this.
Comment 2
Received: 14 May 2020
Commenter: Geraldo Passos
The commenter has declared there is no conflict of interests.
Comment: I agree with the reply from Jadson C. Santos and I would like to add that this preprint was for us to register the idea of ​​trying to make a specific change in the ACE2 molecule that interacts with SARS-CoV-2, but leaving the active enzyme site intact.
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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Comment 3
Received: 14 May 2020
Commenter: Geraldo Passos
The commenter has declared there is no conflict of interests.
Comment: I agree with the reply from Jadson C. Santos and I would like to add that this preprint was for us to register the idea of ​​trying to make a specific change in the ACE2 molecule that interacts with SARS-CoV-2, but leaving the active enzyme site intact.
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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Comment 4
Received: 28 May 2020
The commenter has declared there is no conflict of interests.
Comment: The authors claim that using the CRISPR-Cas9 system to introduce mutations into the ACE2 gene would weaken the ACE2-SARS-CoV2 interaction and reduce the SARS-CoV2 ability to enter the epithelial cell while preserving the biological activity of ACE2
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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Response 1 to Comment 4
Received: 29 May 2020
Commenter: Geraldo Aleixo Passos
The commenter has declared there is no conflict of interests.
Comment: Thank you for your interest.
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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