Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs

Abstract

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.

Keywords: CP: Molecular biology; CRISPR-Cas; CRISPR-Cas12a; Cas12a orthologs; SARS-CoV-2; cCRISPR; clinical diagnostics; combinatorial CRISPR-Cas; gene editing; non-canonical crRNA; nucleic acid detection.

Figure 1.. A survey of in vitro performance of Cas12a proteins

(A) Phylogenetic analysis of 23 Cas12a orthologs. Sequence alignment of Cas12a shows conserved regions of RuvC and Nuc domains. (B) Sequence alignment of mature crRNA from CRISPR direct repeats across all 23 Cas12a orthologs display high similarity and conserved stem-loops. To minimize the number of combinations, identical and/or similar crRNA sequences were clustered together into 9 groups. (C) Percentage of cis-cleavage. Double-stranded GFP fragment was added to the precomplexed Cas12a:crRNA and incubated at 37°C for 30 min followed by gel electrophoresis. The percentage of cleavage of the dsDNA target was analyzed by ImageJ (mean, n = 2 independent experiments). (D) Background corrected fluorescence of 22 functional Cas12a against 9 crRNAs in a combinatorial fashion with TTTTT reporters. The fluorescence signal was collected using the trans-cleavage reporter assay at t = 20 min (mean, n = 3 independent experiments). Canonical crRNAs are highlighted in the boxes with borders. (E) Binary structure of LbCas12a (yellow) in complex with its canonical crRNA (red) (PDB: 5ID6).^, (F) Ternary structure of LbCas12a (yellow) in complex with its canonical crRNA (red) and target DNA (teal) (PDB: 5XUS). Arrow indicates solvent-accessible region in the stem-loop of crRNA (E and F).

Figure 2.. Part of the hairpin loop of the crRNA scaffold is not crucial for cleavage, but sequence changes can enhance the overall performance of Cas12a

RNAfold was used to predict some of the crRNA structures. (A, C, and E) Fluorescence signal of individual truncated crRNAs. (B, D, and F) Fluorescence signal of combined truncated crRNAs. The fluorescence was collected using the trans-cleavage reporter assay at t = 20 min (mean ± SD, n = 3 independent experiments). (G and H) cis-Cleavage assay of all truncated crRNAs from (A) to (F). The cis-cleavage experiment has been repeated with similar results.

Figure 3.. Noncanonical crRNAs increase the thermal stability of some Cas12a

(A–F) Representation of DSF of 3 Cas12a (TsCas12a, ArCas12a, and ErCas12a), where (A), (C), and (E) represent melting curves, and (B), (D), and (F) represent the derivative of fluorescence from (A), (C), and (E), respectively (mean, n = 4, with n = 2 replicates per experiment over 2 independent experiments). For melting curves of the rest of Cas12a, refer to Figure S6. Except for the black curve, which represents apoCas, different color-coded curves refer to the crRNAs complexed with each respective protein. (G) Summary of thermal stability of 22 Cas12a when complexing with 9 different crRNAs. Canonical crRNAs are highlighted in the boxes with borders. The fluorescence signal presented is the mean value with a total n = 4 (n = 2 replicates per experiment over 2 independent experiments).

Figure 4.. Adaptability of noncanonical crRNAs in mammalian cells

(A) Percentage of indels induced by cotransfection of various Cas12a orthologs and crRNAs targeting the CCR5 locus in HEK293T cells (n = 3 biological replicates). Canonical crRNAs are highlighted in the boxes with borders. (B–E) A subset of the percentage of indels data was compared relative to the canonical crRNAs for AsCas12a, ErCas12a, BsCas12a, and FnCas12a, respectively (n = 4). Error bars represent ±1 SD. (F) Pictorial representation of the WT and noncanonical MS2 crRNAs tested. (G) Percentage of indels induced by RNP nucleofection of noncanonical MS2 crRNAs targeting the CCR5 locus in HEK293T cells (n = 3 biological replicates). Error bars represent ±1 SD. All indel percentages were generated with CRISPResso2.

Figure 5.. Validation of patient samples using new Cas12a variants with cCRISPR assay

The 4 proteins, ErCas12a, TsCas12a, BsCas12a, and BoCas12a, with the best combination of crRNA candidates were selected and tested on patient samples. Notably, some noncanonical crRNAs exhibit enhanced trans-cleavage with higher fluorescence signals compared to the WT crRNA. (A–C) CRISPR detection consisting of positive and negative SARS-CoV-2 determination and detection of B.1.1.7 variant via fluorescence reporter assay in nasopharyngeal swabs (A), saliva (B), and tracheal aspirate (C) samples, respectively. The fluorescence shown above was taken at t = 15 min, except for RNase P, taken at t = 30 min. (D–G) Detection performance of ErCas12a (D), BoCas12a (E), BsCas12a (F), and TsCas12a (G) with canonical and noncanonical crRNA. N-gene⁺ clinical samples from nasopharyngeal swabs, saliva samples, and tracheal aspirates were pooled together to evaluate the combinatorial performance of these Cas12a nucleases. A single experiment was performed to simulate the real-world scenario.