Elimination of cis-cleavage in CRISPR Diagnostics for One-Pot Rapid Nucleic Acid Detection

Materials and reagents

Reagents

1. RPA kit and RT-RPA (Weifang Amp-Future Biotech, Changzhou, China, catalog number: WLB8201KIT); contains lyophilized RPA pellets and buffer A; store lyophilized pellets at -20 °C

2. WarmStart^® LAMP Kit (DNA & RNA) (New England Biolabs, catalog number: E1700S); store at -20 °C

3. 10× NEBuffer r2.1 (New England Biolabs, catalog number: B6002S); store at -20 °C

4. SuperScript IV reverse transcriptase (Thermo Fisher Scientific, catalog number: 18090010); store at -20 °C

5. RNase H (New England Biolabs, catalog number: M0297S); the original study used a 50 U/μL working stock; verify the supplied activity concentration; store at -20 °C

6. Proteinase K (Takara, catalog number: 9034); store at -20 °C

7. qPCR Detection kit (GenePharma, Suzhou, China, catalog number: QPG-040); used for orthogonal qPCR/RT-qPCR validation; store at -20 °C

8. FAM–BHQ1 ssDNA FQ reporter, 5′-FAM-TTATT-BHQ1-3′ or 5′-FAM-TTATTATT-3′-BHQ1 [synthesized by Sangon Biotech, Shanghai, China; HOLMES-Fluo ssDNA reporter 1 (FAM) is available from TOLOBIO, catalog number: 31101]; store the 100 μM stock at -20 °C in the dark. Reconstitute lyophilized reporter in nuclease-free water to 100 μM; for a supplied solution, use the stated concentration. Use the same reporter sequence throughout a comparison

9. Scaffold RNA, spacer RNAs, full-length crRNAs, RPA and LAMP primers, and dsDNA targets/gene fragments (synthesized by Sangon Biotech, Shanghai, China; HPLC- or PAGE-purified); dissolve in DEPC-treated water or nuclease-free water and store at -20 °C (RNA stocks may be stored at -80 °C for extended periods); the sequences used in the original study are listed in Supplementary Table S1 of [15]

10. 6×His-pET-28a(+) expression plasmids encoding AsCas12a, LbCas12a, and CtCas12a; constructs are available from Yi Liu

11. Ni-NTA resin (Qiagen, catalog number: 36113)

12. 100 kDa molecular weight cutoff (MWCO) centrifugal filters (Merck Millipore, Amicon Ultra, catalog number: UFC910024)

13. GoldView nucleic acid stain (Yeasen, catalog number: 10201ES)

14. Ultrapure water (18.2 MΩ·cm, from a Milli-Q purification system; used throughout all buffer and solution preparation)

15. Tris base (Trizma^® base) (Sigma-Aldrich, catalog number: T1503); used to prepare 1 M Tris-HCl stocks (pH 7.9 and pH 8.8) and 50× TAE buffer; store at room temperature

16. 1 M Tris-HCl, pH 7.5 (Invitrogen, catalog number: 15567027); alternatively prepared in-house from Tris base adjusted with HCl; store at room temperature

17. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758); used for pH adjustment of Tris-HCl stocks; store at room temperature

18. 5 M NaCl, RNase-free (Invitrogen, catalog number: AM9759); store at room temperature

19. Glycerol, molecular biology grade (Sigma-Aldrich, catalog number: G5516); store at room temperature

20. 1 M MgCl₂, RNase-free (Invitrogen, catalog number: AM9530G); store at room temperature

21. Bovine serum albumin (BSA), 20 mg/mL, molecular biology grade (New England Biolabs, catalog number: B9000S); store at -20 °C

22. Ammonium sulfate [(NH₄)₂SO₄] (Sigma-Aldrich, catalog number: A4418); store at room temperature

23. 1 M MgSO₄ solution, for molecular biology (Sigma-Aldrich, catalog number: M3409); also used for the additional 8 mM MgSO₄ supplementation in the one-pot reaction; store at room temperature

24. Tween^® 20, molecular biology grade (Sigma-Aldrich, catalog number: P9416); store at room temperature

25. Nuclease-free water, not DEPC-treated (Invitrogen, catalog number: AM9937); store at room temperature

26. Glacial acetic acid (Fisher Scientific, catalog number: A38-212); store at room temperature

27. 0.5 M EDTA, pH 8.0 (Invitrogen, catalog number: 15575020); store at room temperature

Storage of critical items: Purified Cas12a proteins are flash-frozen in liquid nitrogen and stored at -80 °C; a quantitative shelf life has not been established. Oligonucleotides (DNA primers, dsDNA targets) are stored at -20 °C; RNA oligonucleotides (scaffold RNA, spacer RNAs, crRNAs) are stored at -20 °C for routine use and at -80 °C for long-term storage. Lyophilized RPA/RT-RPA pellets and kit enzymes are stored at -20 °C.

Solutions

1. Cas12a storage buffer (see Recipes)

2. 10× NEBuffer r2.1 equivalent (see Recipes)

3. 10× LAMP reaction buffer (see Recipes)

4. 10 μM FQ reporter working stock (see Recipes)

5. 50× TAE buffer (see Recipes)

Recipes

1. Cas12a storage buffer (100 mL)

Sterilize by filtration (0.22 μm) and store at 4 °C for up to 3 months.

2. 10× NEBuffer r2.1 equivalent (50 mL)

Adjust to pH 7.9 at 25 °C. Sterilize by filtration (0.22 μm), aliquot, and store at -20 °C for up to 1 year. This is also commercially available (NEB, catalog number: B6002S).

3. 10× LAMP reaction buffer (50 mL)

Adjust to pH 8.8 at 25 °C. Sterilize by filtration (0.22 μm), aliquot, and store at -20 °C for up to 1 year. Note that the additional 8 mM MgSO₄ supplemented in the LAMP/SCas12a one-pot reaction (section E) is provided separately and is not part of this buffer. The nominal total MgSO₄ concentration is therefore 10 mM, including 2 mM from the 1× buffer.

4. 10 μM FQ reporter working stock (100 μL)

Prepare in a low-binding microtube, protect from light, and store at -20 °C in aliquots. Avoid repeated freeze/thaw cycles.

5. 50× TAE buffer (1 L)

Store at room temperature. Dilute to 1× TAE in ultrapure water before use for agarose gel electrophoresis.

Laboratory supplies

1. Pipette tips (filtered) (Thermo, catalog numbers: TF112-1000-Q, T104RS-Q, TF140-200-Q)

2. 0.2 mL PCR tubes (Thermo, catalog number: 431-MIXED-Q)

3. 1.5 mL microtubes (AXYGEN, catalog number: MCT-150-L-C)

Equipment

1. Real-time PCR system (Bio-Rad, model: CFX96 touch, as used in [15]; alternative instruments require verification of thermal control and FAM detection)

2. Microcentrifuge (mySPIN^TM 6 Mini, Thermo, model: 75004061 or equivalent)

3. Thermal cycler (VeritiPro PCR, Thermo, model: A48141)

4. Vortex mixer (Eppendorf mixer, Eppendorf, model: 5382000074)

5. Variable volume pipettes (P10, P200, P1000)

6. Freezer (-80 °C) (Thermo, model: 905)

7. Standard laboratory equipment (analytical balance, pH meter, etc.)

Software and datasets

1. CFX Maestro software (Bio-Rad, version 2.3 or compatible; supplied with the CFX96 Touch system; requires a license bundled with the instrument) for real-time fluorescence acquisition and baseline analysis

2. NUPACK (http://www.nupack.org/; accessed January 12, 2026; free for academic use) for screening spacer RNA sequences for minimal secondary structure. Free alternatives: RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) and Mfold (http://unafold.rna.albany.edu/?q=mfold)

3. AlphaFold3 Server (https://alphafoldserver.com/; free for non-commercial use; optional) for predictive structural modeling of the Cas12a/split crRNA/DNA complex

4. GraphPad Prism (version 8 or later; commercial license required) for plotting fluorescence kinetics, bar charts, and heatmaps. Alternatives: Microsoft Excel (commercial), R/RStudio, or Python (matplotlib). Record the actual software versions and prediction settings used; the listed versions are reference implementations

5. Oligonucleotide sequences (scaffold RNA, spacer RNAs, primers, and targets) used in the original study are listed in Supplementary Table S1 of Yin et al. [15] (Nucleic Acids Research, DOI: 10.1093/nar/gkag267). The complete source list is accessible through the Supplementary data link at https://doi.org/10.1093/nar/gkag267

Procedure