Author contributions K.C. and L.P. conceived the project, led the study, and wrote the software. K.C. analyzed experimental data. All authors contributed input on measurement and visualization of genome editing outcomes and provided input on the manuscript. Competing Interests At the time of manuscript preparation, J.M.G. was a consultant for Beam Therapeutics, and now is employed by Beam Therapeutics. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Endcadia, EpiLogic Therapeutics, Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.M.G. and J.K.J. are co-inventors on patents and patent applications that describe gene editing technologies. D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants and Beam Therapeutics, companies that use genome editing. Code availability.

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K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.