PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/μL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids

Additional file 2: Figure S1. of CRISPR/Cas9-mediated viral interference in plants

Dot blot analysis of the TYLCV genome accumulation in NBCas9OE. Figure S2 CRISPR/Cas9-mediated virus interference in TYLCV sap inoculated plants. Figure S3. Targeting of CP region of TYLCV by CRISPR/Cas9. Figure S4. RCA analysis of the TYLCV genome accumulation. Figure S5. DNA blot analysis of the TYLCV genome accumulation. Figure S6. Reduction of TYLCV symptoms on NB-Cas9OE plants expressing IR-sgRNA. Figure S7. Reduction of TYLCV symptoms in NB-Cas9OE plants expressing CP-gRNA or RCRII-gRNA. Figure S8. Reduction of TYLCV symptoms in NB-Cas9OE plants coexpressing IR-sgRNA and CP-sgRNA. Figure S9. Restriction enzyme recognition site loss analysis from multiplexed targeting of IR and CP sequences. Figure S10. Alignment of the Sanger sequence reads of IR and CP regions of TYLCV from multiplexed targeting of IR and CP sequences. Figure S11. Recovery of TYLCV symptoms in NB-Cas9OE plants expressing IR-CP-gRNA. Figure S12. Southern blot analysis for the TYLCV genome accumulation. (PDF 3075 kb

Additional file 2: Table S1. of Herboxidiene triggers splicing repression and abiotic stress responses in plants

includes information for primers used in this paper. (XLSX 14 kb

Additional file 1: Table S1. of CRISPR/Cas9-mediated viral interference in plants

Primers used in this study. Table S2 Summary of different sgRNA used for targeting of TYLCV genome. (PDF 3026 kb

Additional file 4: of CRISPR/Cas9-mediated viral interference in plants

Supplementary sequences and maps. Supplemental sequence 1. TYLCV 2.3 genome sequence and map. Supplemental sequence 2. IR-gRNA (TYLCV) sequence and map. Supplemental sequence 3. CP-gRNA (TYLCV) sequence and map. Supplemental sequence 4. RCRII-gRNA (TYLCV) sequence and map. Supplementary sequence 5. IR-gRNA (BCTV Worland) sequence and map. Supplementary sequence 6. RCRII-gRNA (BCTV Worland) sequence and map. Supplementary sequence 7. BCTV (Worland) sequence and map. Supplementary sequence 8. MeMV sequence and map. Supplementary sequence 9. Non-specific-sgRNA sequence and map. (PDF 3031 kb

Additional file 3: of CRISPR/Cas9-mediated viral interference in plants

Supplementary methods. (PDF 71 kb

Additional file 1: of Herboxidiene triggers splicing repression and abiotic stress responses in plants

This file contains all supporting Supplementary Figures. (PDF 861 kb

Additional file 2: Sequence S1: of RNA virus interference via CRISPR/Cas13a system in plants

pCas13a amino acid sequence (3xHA-pCas13a-nls). Sequence S2: pCas13a full-length plant codon optimized DNA sequence (3x-HA-pCas13a-nls) Map S1: pCas13a sequence in pK2GW7-pCas13a (pK2GW7-3xHA-pCas13a-nls). Sequence S3: TuMV-GFP full-length sequence with target sequences in different colors. Map S2: Map of TuMV-GFP. Sequence S4: Cas13a-repeat-cRNA-TuMV-GFP-GFP-target 1 sequence (A), map (B) and its complex with targeting region in TuMV GFP genomic region (C). Sequence S5: Cas13a-repeat-cRNA-TuMV-GFP-GFP-T2 sequence (A), map (B), and its complex with targeting area in TuMV GFP genomic region (C). Sequence S6: Cas13a-repeat-cRNA-TuMV-GFP-HC-Pro-T1 sequence (A), map (B), and its complex with HC-pro targeting region in TuMV (C). Sequence S7: Cas13a-repeat-cRNA-TuMV-GFP-Cp-Pro-T1 sequence (A), map (B), and its complex with Capsid protein targeting region in TuMV. (DOCX 414 kb

Additional file 3: Table S1: of RNA virus interference via CRISPR/Cas13a system in plants

TuMV genome description. Table S2: Primers used in this study. (DOCX 21 kb

Additional file 1: Figure S1. of RNA virus interference via CRISPR/Cas13a system in plants

Confirmation of pCas13a expression in planta. Figure S2. pCas13a-mediated interference with TuMV-GFP in planta. Figure S3. GFP quantification of TuMV-GFP interference in transgenic pCas13a-OE plants. (PDF 1393 kb