Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-ß-lactamases.

Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-ß-lactamases (MßLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MßLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MßLs. This common mechanism open avenues for rationally designed inhibitors of all MßLs, notwithstanding the profound differences between these enzymes' active site structure, ß-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-ß-lactamases (MßLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MßLs share the same reaction mechanism, suggesting new strategies for drug design.