Structure-Guided design of Cas12a variants improves detection of nucleic acids

Figures

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  Fig. 1. One-pot detection using LbCas12a mutants. (A) Up: the schematic diagram of the Cas12a K595A promoting the one-pot reaction. Bottom: cis-cleavage (specific cleavage) and trans-cleavage (non-specific cleavage) of LbCas12a. ‘F’ and ‘Q’ represent fluorophore and quencher, respectively. ‘T’ indicates the single-stranded DNA fluorophore-quencher reporter sequence of TTATT. (B) The key amino acids labeled in the ternary complex structure of LbCas12a-crRNA-dsDNA (PDB number is 5XUS). (C) The fluorescence signal generated by the Cas12a mutants and WT in one-pot reactions. (D) Comparison of one-pot methods mediated by Cas12a-WT protein, K595A mutant, and suboptimal PAM-mediated approaches targeting the SARS-CoV-2 Orf1ab gene and E gene.

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  Fig. 2. The performance of LbCas12a WT and K595A mutant for detection. (A)-(F) The fluorescence curves ((A)–(B)), sensitivities ((C)–(D)) and reproducibilities ((E)–(F)) of one-pot reactions mediated by LbCas12a WT or K595A mutant. Mean ± s.d. of n = 4 technical replicates for (C) and (D), and n = 10 for (E) and (F). The SARS-CoV-2 E gene and Orf1ab gene were used as substrates.

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  Fig. 3. Kinetics of LbCas12a WT and K595A mutant. (A)-(D)Cis-cleavage activity of LbCas12a WT and K595A mutant on E gene plasmid of 3314 bp and Orf1ab gene dsDNA of 461 bp. ‘S’ indicates plasmid substrates ((A), (B)) or dsDNA substrates ((C), (D)); ‘P’ indicates cleaved products; ‘N’ indicates nick, ‘L’ indicates linear DNA. The products of Orf1ab gene dsDNA are 242 bp and 219 bp. (E)-(F) Accumulation of RPA amplicons in one-pot reaction mediated by LbCas12a WT or K595A mutant at 37 °C for 0–20 min.

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  Fig. 4. Detection of ASFV using K595A mutant. (A) Limits of detection using LbCas12a K595A-mediated one-pot reaction. Mean ± s.d. of n = 4 technical replicates for ASFV POL dsDNA, and n = 2 for NC. (B) Fluorescence values of LbCas12a K595A-mediated one-pot reaction for detecting ASFV samples.

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  Fig. 5. Sequence and structure of Cas12a orthologs. (A) Sequence alignment of LbCas12a, AsCas12a and FnCas12a. (B)-(C) Hydrogen-bond interactions between PAM and the lysine residues of AsCas12a (B) and FnCas12a (C). The structures of AsCas12a (PDB number: 5B43) and FnCas12a (PDB number: 6I1K) were generated by PyMOL software.