Procedure

DNase I treatment (Before clean-up)

1. IMPORTANT: Prior to use, reconstitute the lyophilized DNase I as indicated on the vial. Store frozen aliquots.
2. For each sample to be treated, prepare DNase I reaction mix in an RNase-free tube (not provided). Mix well by gentle inversion, once the following components added (volume of components to add to the RNA sample can be readjusted according to the sample's volume)

Product	Volume
RNA sample (≤10 μg) volume adjusted with water or TE buffer	40 μl
DNase I	5 μl
DNA Digestion Buffer	5 μl
Total volume	50 μl

3. Incubate at room temperature (20-30ºC) for 15 minutes.

Buffer preparaiton

1. Before starting, add 48 ml 100% ethanol (52ml 95% ethanol) to the 12 ml RNA Wash Buffer concentrate(R1013, R1015) or 96 ml 100% ethanol (104ml of 95% ethanol) to the 24 ml RNA Wash Buffer concentrate (R1014, R1016).

Wash

All centrifugation steps should be performed at 10,000 –16,000 x g. RNA species ≥17 nt will be recovered.

1. Add 2 volumes RNA Binding Buffer to each sample and mix. Example: Mix 100 μl buffer and 50 μl sample.
2. Add an equal volume of ethanol (95-100%) and mix. Example: Add 150 μl ethanol.
3. Transfer the sample to the Zymo-Spin™IC Column in a Collection Tube and centrifuge for 30 seconds. Discard the flow-through.
4. Add 400 μl RNA Prep Buffer to the column and centrifuge for 30 seconds. Discard the flow-through.
5. Add 700μl RNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through.
6. Add 400 μl RNA Wash Buffer to the column and centrifuge for 2 minutes to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube (not provided in the kit).

Elution

Add 15μl DNase/RNase-Free Water directly to the column matrix and centrifuge for 30 seconds. (Alternatively, for highly concentratedRNA use ≥ 6μl elution).The eluted RNA can be used immediately or stored at -70°C.

References

1. Promega: "Synthesis of Nonlabeled RNA" protocol, https://www.promega.com/-/media/files/resources/protocols/product-information-sheets/n/t7-rna-polymerase-protocol.pdf
2. ZYMO Research: RNA Clean & Concentrator™-5, Instruction Manual, https://www.zymoresearch.eu/media/amasty/amfile/attach/_R1013_R1014_R1015_R1016_RNA_Clean_Concentrator-5_ver2.2.1.pdf

crRNA Transcription using T7 RNA Polymerase (Promega)

Introduction

In this part we're going to transcribe the CRISPR RNA (crRNA) required for the CRISPR-Cas12a assay using the isothermal T7 RNA polymerase. This enzyme will only transcribe DNA downstream of a double-stranded T7 promoter (in the 5' to 3' direction), thus we had to fuse the single-stranded DNA (ssDNA) coding sequence (CDS, flanked on its 3' end with the 3' to 5' strand of T7 promoter) with a primer (T7 primer, complementary 5' to 3' ) in order to constitute the promoter and obtain efficient transcription. We initially followed the Annealing oligonucleotides protocol in order to anneal the T7 primer to the ssDNA which will constitute our DNA template. This is based on Promega's protocol: "Synthesis of Nonlabeled RNA" [1]

Materials

• Transcription Optimized 5X Buffer
• DTT, 100mM
• Recombinant RNasin® Ribonuclease inhibitor
• rATP, rGTP, rUTP, rCTP
• DNA template* (annealed ssDNA + primer) in water ( orTE buffer at 2–5μg)
• T7 RNA polymerase (Phage RNA polymerase)
• Nuclease-Free Water

Procedure

1. Make the rNTP mix as following

Products	Concentration
rATP	2.5 mM
rGTP	2.5 mM
rUTP	2.5 mM
rCTP	2.5 mM
In nuclease free water

2. In a microcentrifuge tube, add the following reagents at room temperature in the order listed

Materials	quantity
Transcription Optimized 5X Buffer	20μl
DTT, 100mM	10μl
Recombinant RNasin® Ribonuclease Inhibitor	100 units
rNTP mix	20μl
DNA template, linearized (in water or TE buffer at 2–5μg, i.e. ~100 µM concentrated)*	2μl
Phage RNA polymerase	40 units
Nuclease-Free Water to final volume of	100μl

3. Incubate for 2 hours at 37°C.
4. Purify the sample following the RNA purification protocol.

References

1. Promega: "Synthesis of Nonlabeled RNA" protocol, https://www.promega.com/-/media/files/resources/protocols/product-information-sheets/n/t7-rna-polymerase-protocol.pdf

Fluorophore-Quencher reporter Cas12a assay

Introduction

This assay's purpose is to detect a specific DNA sequence (the activator) using the CRISPR/Cas12a system. Cas12a's feature is to cleave any ssDNA in the sample once it has found its target. We used this to our advantage and used a single-stranded fluorophore-quencher reporter (DNaseAlert) to be able to quantify our sequence of interest using a plate reader. This protocol is based on and optimized from the LbCas12a collateral detection protocol ("Fluorophore quencher (FQ)-labeled reporter assays")[1]

Materials

• EnGen® Lba Cas12a (Cpf1) 1 μM
• Purified crRNA from DNA sequence 1μM
• 10X Binding buffer (200 mM Tris-HCl, pH 7.5, 1 M KCl, 50 mM MgCl2, 50% glycerol, 500 μg/ml heparin, 1mM DTT)
• DNaseAlert™ (IDT) 1 µM
• Nuclease-Free Water
• 6 of small pcr tubes (0.2 ml) for your master mix and samples
• DNase I enzyme (Zymo)
• 384 well Plate
• Multi Well Plate Sealing Films

Procedure

1. Preparation of Cas12a master mix

	LbCas12a concentration	gRNA concentration	Activator concentraion	DNaseAlert concentration	Buffer	Dnase I
FQ1	62.5 nM	75 nM	Variable	160 nM	1X Binding	-
Negative control	62.5 nM	75 nM	-	160 nM	1X Binding	-
Blank	-	-	-	-	1X Binding	-
Positive control	-	-	-	160 nM	1X Binding	2.4 μl

• Add the following materials except the DNaseAlert and activator into a 0.2 ml tubes. Incubate at 37°C for 30 min, then add the rest of the components.

Cmponents	FQ1	Negative control	Blank	Positive Control
10X Binding Buffer	6.6	6.6	6.6	6.6
Cas12 [1µM]	3.75	3.75	-	-
crRNA [1µM]	4.5	4.5	-	-
Nuclease Free Water	29.6	35.6	53.4	41.4
Incubation 30 minutes at 37°C
DNase Alert	9.6	9.6	-	9.6
Activator	6	-	-	-
DNase I	-	-	-	2.4
Final volume (µL)	60	60	60	60

2. Prepare the Optical Plate
• Load 24 µl of each corresponding tubes into a 384 opti plate (in duplicate) in the following order:

Negative	FQ1	blank	Positive control
Negative	FQ2	blank	Positive control

• Stick an adhesive film on the top of the plate.
3. Put it in the plate reader. Set up the device: at 535 nm excitation and 590 nm emission, 37℃ and take the measurements every 20 seconds for 180 repeats.
4. Plot a graph of the fluorescence as a function of time (minutes), taking the average of the fluorescence obtained for each well.

References

• [1]Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439.

Oligonucleotides/gBlocks Resuspension and Storage (IDT)

Introduction

Recommendations on how to resuspend and store oligos or/and gBlocks gene fragments (IDT) once shipped. Check IDT's page: "My oligos have arrived: Now what?"1 for more details.

Materials

• Oligos or gBlocks gene fragments
• Nuclease-free water/TE buffer

Procedure

1. Briefly centrifuge the tubes before opening them
2. the oligos should be resuspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). Nuclease-free water (pH 7.0) may be used alternatively. However, use of HPLC- or molecular biology–grade water is preferable. CAUTION: Nuclease-free water will not modulate pH over time as will TE buffer.
3. Standard recommendation: Resuspend oligos to a 100 µM stock concentration, The volume of TE buffer required to achieve a 100 µM stock is easily determined by multiplying the number of nanomoles (nmol) listed for a particular oligo by a factor of 10, and then resuspending the dry DNA in that same number of microliters of TE buffer. For example, if the oligo specification sheet states that 20.3 nmol of oligo were delivered, add 203 µL TE buffer to obtain a 100 µM stock solution. This stock solution can be diluted as needed into appropriate working solutions.
4. For oligos that are harder to resuspend, and for which one might observe residual precipitate present following resuspension, the oligo should be heated at 55°C for 1–5 minutes, vortexed thoroughly, and then briefly centrifuged.

Storage

• Store your resuspended oligonucleotides at -20°C (stable for at least 24 months when either dried down, or resuspended in TE buffer or nuclease-free water).

References

• [1] Integrated DNA Technologies (IDT): "My oligos have arrived: Now what?", https://eu.idtdna.com/pages/education/decoded/article/my-oligos-have-arrived-now-what-; "Tips for resuspending and diluting your oligonucleotides", https://eu.idtdna.com/pages/education/decoded/article/tips-for-resuspending-and-diluting-your-oligonucleotides

PCR amplification in Plasma

Introduction

This is a protocol on how to amplify DNA fragments in plasma. Since we are going to target specific DNA fragments with our CRSPR-cas12a assay following this PCR we will add ourselves the oligos in our sample. If you want to amplify DNA that is already contained in your sample skip the additon of DNA in plasma. This protocol is based on the one found in PCR protocols by Professor Kenji Abe1.

Materials

• DNA template
• PCR primers
• dNTPs (10 mM)
• Nuclease-Free water
• 5X Phusion HF buffer
• Phusion DNA polymerase
• Human Blood Plasma
• 10x Phosphate-buffered Saline (PBS)
• Thermal cycler

Procedure

1. Plasma samples will be dilutated 1:5 in 1x PBS but as not to dilute the plasma too much we will proceed by adding the DNA template in PBS in the following way
2. Make a mastermix of 1 part plasma (4μl) in 3 part PBS diluted as following: 1.6μl 10X PBS in 10.4μl NFW

We want the PBS to be 1X in 4 part of the sample (16μl) but one part is used to put the templated. Once we add the last part the PBS will become 1X.

3. Put 8μl of the master mix in two different tubes and make the two following samples:

Components	Sample A	Negative control
Template	1µl	-
Nuclease free water	1μl	2µl
Total	2μl	2µl

4. The diluted plasma sample are heated for 3 min at 95℃ then cooled rapidly on ice for 3 to 5 min.
5. Make the following MasterMix

Components	A	Negative control	MasterMix 2.5x	Final concentration
NFW	28.5	28.5	71.25	-
5X Phusion HF buffer	10	10	25	1X
10 mM dNTPs	1	1	2.5	200 µM
Forward primer (10 μM)	2.5	2.5	6.25	0.5 µM
Reverse primer (10μM)	2.5	2.5	6.25	0.5 µM
From Master mix	44.5	44.5	111.25	-
Sample A	-	5	-	-
Negative control	5	-	-	Variable
Phusion DNA Polymerase	0.5	0.5	-	1.0 units/50 µl PCR
Total volume	50	50	-	-

6. Gently mix the reaction. Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 °C and begin thermocycling:

Temperature	Time	Number of cycles
98 °C	30 sec (Initial Denaturation)	1
98 °C	10 sec (denaturation)	30 cycles
Variable	30 sec (primer annealing)	30 cycles
72 °C	30 sec (extension)	30 cycles
72 °C	10 min (Final Extension)	1
4 °C		infinite

References

1. 1 Abe, Kenji. « Direct PCR from Serum ». PCR Protocols, edited by John M. S. Bartlett and David Stirling, Humana Press, 2003, p. 161‑66. Springer Link, doi:10.1385/1-59259-384-4:161

PCR using Phusion® High-Fidelity DNA Polymerase

Introduction

A standard PCR protocol using Phusion high fidelity DNA polymerase. Mainly used for amplifying the junction/point mutated fragments we aim to detect using CRISPR-Cas12a. The protocol is based on NEB PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530)1.

Materials

• DNA template
• Forward primer
• Reverse primer
• dNTPs, 10 mM
• Nuclease-Free water
• 5X Phusion high fidelity (HF) buffer
• Phusion DNA polymerase (ThermoFisher, 2 U/µl)
• Thermal cycler

Procedure

1. The thermal cycler (PCR machine) should be programmed as following. IMPORTANT: Annealing temperature may vary depending on the primer's melting temperature (Tm).

Step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	-
Denaturation	98°C	10 sec	35
Primer annealing	45-72°C (depending on primers' Tm)	30 sec	35
Extension	72°C	30 sec	35
Final extension	72°C	10 min	-
-	4°C	-	-

2. For one sample, add the following amounts of products. If working with several samples, a master mix containing the elements that do not change between each sample (water, buffer, dNTPs or/and primers/DNA) can be prepared. However, ALWAYS add the enzyme in the end.

Component	Volume µl	Final concentration
Nuclease-free water	Up to 50 µl	-
5X Phusion HF buffer	10	1X
10 mM dNTPs	1	200 µM
10 µM Forward primer	2.5	0.5 µM
10 µM Reverse primer	2.5	0.5 µM
Template DNA	variable	Up to 250 ng
Phusion DNA Polymerase	0.5	1.0 units/50 µl PCR
Total volume	50	-

3. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Transfer PCR tubes from ice to a PCR machine (programmed previously) and begin thermocycling.
4. Control your samples by doing an agarose gel electrophoresis following the Agarose gel electrophoresis protocol.

References

• 1. New England BioLabs (NEB). PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530): https://international.neb.com/Protocols/0001/01/01/pcr-protocol-m0530