LwCas13a detection reactions were performed as previously described with several modifications. We chose LwCas13a rather than Leptotrichia shahii Cas13a because LwCas13a has been demonstrated to have higher RNA-guided RNase activity than LsCas13a and is better characterized in the context of SHERLOCK assays [11] . Each reaction was performed using 25 μL total volume and contained 45 nM LwCas13, 100-500 ng crRNA, 125 nM fluorescent RNA reporter (RNAse Alert v2, ThermoFisher), 0·625 μL murine RNase inhibitor (New England Biolabs), 31·25 ng background RNA (isolated from Burkitt's Lymphoma (Raji), ThermoFisher), 1 mM rNTP mix (New England Biolabs), 0·75 μL T7 polymerase (New England Biolabs), 3mM MgCl2, and 1·25 uL of RPA product in nuclease assay buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCl2, pH 6·8) [10] (link). Detection reactions were conducted in 96-well black half-area microplates (PerkinElmer, Waltham, MA) and sealed with MicroAmp optical adhesive film (ThermoFisher). Detection reactions were incubated for 3 hours at 37°C on a VICTOR Nivo fluorescent plate reader (PerkinElmer) with fluorescence measurements taken every 5 minutes. Background subtracted fluorescence for each sample was calculated by subtracting the average fluorescence intensity of the human gDNA controls at 180 minutes of the human gDNA control from each sample.
Rntp mix
Manufactured by New England Biolabs
The RNTP mix is a solution containing a balanced mixture of ribonucleotide triphosphates (rNTPs) for use in reverse transcription and other RNA-based applications. The product provides the essential components required for in vitro RNA synthesis and amplification.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
T7 polymerase, by New England Biolabs (1 mentions)
Microamp optical adhesive film, by Thermo Fisher Scientific (1 mentions)
Rnase alert v2, by Thermo Fisher Scientific (1 mentions)
Murine rnase inhibitor, by New England Biolabs (1 mentions)
T7 polymerase, by Beyotime (1 mentions)
Rox reference dye, by Thermo Fisher Scientific (1 mentions)
Lwacas13a, by GenScript (1 mentions)
Pyrophosphatase enzymes, by Aldevron (1 mentions)
Guanylyl transferase, by Aldevron (1 mentions)
Ribogreen assay, by Thermo Fisher Scientific (1 mentions)
Capto core 700 resin, by GE Healthcare (1 mentions)
Dnase 1, by Aldevron (1 mentions)
T7 polymerase, by Aldevron (1 mentions)
Rnase inhibitor, by Aldevron (1 mentions)
S adenosyl methionine, by Aldevron (1 mentions)
4 protocols using rntp mix
1
Optimized LwCas13a CRISPR-based Detection Assay
2
RNA Interference for vab-15 and ref-2
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Full-length cDNAs of vab-15 and ref-2 were cloned to pL4440. In vitro transcription (IVT) template contain dual opposing T7 promoters were generated by PCR with primer sequences: 5′-gttttcccagtcacgacgtt-3′ and 5′-cgaggaagcaacctggctta-3′. Large scale IVT were performed with 3 μL unpurified PCR product, 2 μL T7 Polymerase (Beyotime Biotechnology), 2 μL rNTP mix (NEB), 1 μL RNAse Inhibitor (Vazyme or Takara) and 2 μL homemade T7 Transcription 5X buffer (400 mM HEPES-KOH pH 7.5, 120 mM MgCl2, 10 mM spermidine and 200 mM DTT) at 37 °C for 4.5 h. After IVT, dsRNA was purified with LiCl precipitation and RNA concentration was quantified by agarose gel electrophoresis and nanodrop. dsRNA was injected into young adult hermaphrotides with concentration at 1000 ng/μL for ref-2 and with concentration at 300 ng/μL for vab-15 respectively. Strain thuSi553[Pclec-252::neonGreen::H2B::let-858 3′UTR];stIs10631[ref-2a::H1::mCherry] were used for G2 phenotyping and juIs76[Punc-25::GFP];stIs10631[ref-2a::H1::mCherry] for W phenotyping (Supplementary Data 7 ).
3
CRISPR-Cas13a Assay Preparation
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Assay mixes were prepared in 16 μL volumes for each unique crRNA target consisting of 1 μL of 50 U/μL NxGen T7 Polymerase (Biosearch Technologies 30223–1), 2 μL of 800 nM LwaCas13a (Genscript), 1 μL of a 1.6 μM target-specific Cas13 crRNA, 8 μL of 2X Fluidigm Assay Loading Buffer, and 4 μL of nuclease-free water. 1.5X sample premixes were created with 2.4 μL of 10X T7 Buffer (Biosearch Technologies F88905–1, Hoddesdon, United Kingdom), 3.2 μL of 7.5X sample buffer, 0.2 μL of murine RNase inhibitor (NEB M0314S, Ipswich, MA), 0.96 μL of 25 mM rNTP mix (NEB N0466S, Ipswich, MA), 0.12 μL of 100 μM 6U FAM reporter (IDT), 0.48 μL of 50X ROX reference dye (Thermo Fisher Scientific 12223012, Waltham, MA), 1.20 μL of 20X GE buffer, and 7.44 μL of nuclease-free water. 7.5X Sample Buffer solutions were made in 5 mL aliquots with 375 μL of Tris-HCl, 75 μL of 5M NaCl, 37.5 μL of 1M MgCl2, 750 mg of PEG-8000, and 4512.5 μL of nuclease-free water. Sample premixes were added to pre-amplified sample mixtures at a 2:1 ratio of premix to sample.
4
In Vitro Transcription of saRNA
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Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using NotIlinearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified using Capto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). Terminal filtration of the saRNA material was done using a 0.22 µm PES filter, and the saRNA materials were stored at -80°C until use/complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity, and RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).
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