TITLE: sgRNA Synthesis &amp; CRISPR-Cas9 Synthesis
AUTHORS: priceal  , ekmatozel  , parzialest  

[DESCRIPTION]
A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency—in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA. 



[STEPS]
SECTION: Preparation of sgRNA
14. Gather the materials used in Step 14 (See Materials section). 
SECTION: Preparation of sgRNA
15. Thaw EnGen 2X sgRNA Reaction Mix, S. pyogenes, and the customer-supplied target-specific oligo (1 µM). Mix and pulse each component in microfuge prior to use for 10 seconds. Store enzyme mix on ice.
SECTION: Preparation of sgRNA
16. Assemble the reaction at room temperature in the order listed below. Add the enzyme last to each reaction.

 

SECTION: Preparation of sgRNA
18. Transfer reaction to ice. 
SECTION: Preparation of sgRNA
17. Mix thoroughly and pulse-spin in a microfuge for 10 seconds. Incubate at 37°C for 30 minutes.
SECTION: Preparation of sgRNA
19. For DNase treatment, bring volume to 50 µL by adding 30 µL of nuclease-free water. Add 2 µL of DNase I, mix and incubate at 37°C for 15 minutes.
SECTION: Preparation of sgRNA
20. Gather the materials used in Step 20 (See Materials section). 
SECTION: Preparation of sgRNA
21. Use Monarch RNA Cleanup Kit (50 µg) or similar RNA purification kit. From here, follow the protocol printed inside of the kit. Ensure that ethanol has been added to the correct buffer. 


SECTION: Activation of dCas9 and Binding to DNA
22. Gather the materials used in Step 22 (See Materials section). 
SECTION: Activation of dCas9 and Binding to DNA
24. Assemble the reaction below, in order, at room temperature. The final concentration of both the dCas9 and sgRNA should be 60 nM.

 

SECTION: Activation of dCas9 and Binding to DNA
25. Mix thoroughly by pipetting mixture up and down gently. Pulse-spin in a microfuge. Incubate at 25°C for 10 minutes.
SECTION: Activation of dCas9 and Binding to DNA
27. Mix thoroughly by pipetting mixture up and down gently. Pulse-spin in a microfuge. Incubate at 37°C for 15 minutes.
SECTION: Activation of dCas9 and Binding to DNA
28. Transfer reaction to ice. 
SECTION: Activation of dCas9 and Binding to DNA
29. Add enough glycerol to tube so that it is equal to total reaction volume. Mix thoroughly by pipetting mixture up and down.
    - Ex. Add 28.12 µL glycerol to 28.12 µL reaction mixture
SECTION: Activation of dCas9 and Binding to DNA
26. Add enough DNA to reaction tube so that the final concentration of DNA is 3 nM. The ratio of dCas9:sgRNA:DNA should be 20:20:1. 
    - Ex. Add 1.12 µL of 75 nM stock DNA to reaction tube
SECTION: PCR Preparation
1. Gather the materials used in Step 1 (See Materials section). Thaw components before use. Keep all on ice.
2. Dilute the primers for (See Materials) to 10 uM before using.
SECTION: PCR reaction
3. Combine the components from Step 1 into PCR tube(s) in order, for the desired final volume. Use Table 1 for reference. 

 

5. Advance to purification steps
SECTION: PCR reaction
4. Perform PCR using Table 2 for parameters

 


SECTION: Purification of PCR Product
6. Gather the materials used in Step 6 (See Materials section). 
SECTION: Purification of PCR Product
7. Perform Qiagen PCR purification kit by following the printed protocol inside the kit. Make sure pure ethanol (200 Proof) has been added to the correct buffer.

For 1x volume (50 uL total), you must use a blank centrifuge tube with water to counterbalance the spin column.  

For 2x volume (100 uL total), use two spin columns and add 50 uL to each column.  This will produce the same concentration as 1x protocol, but twice the volume (2x the amount of DNA).

For 4x volume (200 uL total), use two spin columns and add 100 uL (two PCR tubes) to each column.  This will produce twice the concentration of the 1x protocol, and twice the volume (4x the amount of DNA).

Store on ice after purification.
SECTION: Determination of DNA Concentration
8. Gather the materials used in Step 8 (See Materials section). 
SECTION: Determination of DNA Concentration
11. Collect concentrations 2 times each.
SECTION: Determination of DNA Concentration
10. Blank with Buffer EB or dd H2O, depending on how you elute in your PCR purification kit. 
SECTION: Determination of DNA Concentration
9. Use a nanodrop or similar UV absorbance instrument for measurements.
SECTION: Determination of DNA Concentration
12. Take the average of your 2 readings for each DNA sample. 
SECTION: Determination of DNA Concentration
13. Convert ng/uL to nM using the formula below.  Record both ng/uL and nM numbers. 

Concentration [nM] = 1.62 × Concentration [ng/uL]  
SECTION: Activation of dCas9 and Binding to DNA
23. Thaw NEBuffer 3.1, 500 nM sgRNA, and 1 µM dCas9. Mix and pulse each component in microfuge prior to use. Store tubes on ice.
SECTION: Activation of dCas9 and Binding to DNA
30. Roadblocks are now bound to programmed sites on DNA. Store in freezer.