Easy Cloning: Protocol for sgRNA Insertion

talimur
Jul 4
5 min read

Authors: Abu Musa Md Talimur Reza

This protocol describes the step-by-step procedure for cloning single-guide RNA (sgRNA) into a plasmid vector using restriction enzyme-based cloning. The steps include oligo preparation, vector digestion, ligation, bacterial transformation, and insert verification.

Requirements list:

Oligos for insert preparation (normal desalt oligos are enough, no need for specially purified oligos)
U6 forward primer (for sequencing)
Cloning vector (Cas9 expressing vector with sgRNA cloning site or independent sgRNA cloning vector)
Restriction enzyme for cloning: (usually BbsI or BsmBI: confirm with your vector)
Restriction enzyme for insert check (check your plasmid map: usually two different enzymes unless there is two recognition site for one enzyme)
Restriction enzyme buffers (should be compatible with your enzymes)
T4 DNA ligase enzyme
T4 DNA ligase buffer
Agar plate with selection antibiotic
LB broth
Selection antibiotic (usually ampicillin, but may differ, confirm with your vector)
Competent cells (Stbl3, Stbl4 or NEB stable bacteria strain should be used)
Heat block (with cooling capacity is desired)
Ice water bath (prepare in a beaker using ice and water: ice-water)
Spreader (glass rod bent into an "L" or "hockey stick)
Shaking incubator
Plasmid Mini-prep Kit (commercial or lab-made)
Conical flask
20 mL autoclaved glass tubes (15 mL plastic tubes are also fine)
Agarose
DNA binding dye (use Safe dye, avoid Ethidium Bromide or EtBr)
Electrophoresis apparatus (Horizontal) with power supply
TAE or TBE buffer (TAE is cost-efficient)
Transilluminator (ideally Blue Light, avoid UV-illuminator unless it is integrated with a protected Gel-doc system)
1.5 mL Eppendorf tubes
2 mL Eppendorf tubes
Nuclease free water

Protocol:

1. Preparation of sgRNA Oligos

Spin the primer-containing vial.
Dilute each oligo to a concentration of 100 pmol/µL using autoclaved double-distilled water (MilliQ) or TE buffer.

Note: if primers are delivered in liquid form, adjust the volume to reach the required concentration.

2. Annealing of the sgRNAs

Mix 10 µL sgRNA and 10 µL reverse complement oligo in a 1.5 mL microcentrifuge tube.
Heat the mixture at 100°C for 5 minutes using a heat block.
Allow the tube to cool at room temperature for at least 1 hour.

Note: Heating denatures the oligos, and subsequent cooling enables hybridization to form the double-stranded insert.

3. Digestion of the Plasmid Vector

Add the following into a 1.5 mL tube:
- ~200 ng of plasmid DNA
- 5 µL of 10X digestion buffer
- 1 µL of restriction enzyme
- MilliQ water to bring the final volume to 50 µL
Incubate at 37°C for 1–2 hours or as recommended for the enzyme.
Heat at 70°C for 10 minutes to deactivate the restriction enzyme.

Note 1: The buffer should match the enzyme’s optimal conditions. FastDigest enzymes must be used with FastDigest buffer.

Note 2: When using multiple enzymes, add 1 µL of each.

Note 3: For sgRNA cloning, usually the vectors are designed for Esp3I (BsmBI) or BbsI (BpiI). These are Type IIS enzymes that cut outside its recognition site, and requires two recognition sites. Please check your vector for the correct enzyme.

Note 4: You may prepare a 20 µL reaction mixture without any issue. You can also digest a higher amount of plasmid (up to ~1 µg). In that case, you may need to digest for a longer period of time, and need to make sure that you are not crossing the time limit of star activity (non-specific cleavage) of your enzyme.

4. Purification of Digested Plasmid

Add 500 µL binding buffer or follow the manufacturer’s instructions.
Purify the digested plasmid using a PCR purification kit.
Elute the plasmid DNA in 30 µL of elution buffer or MilliQ water.

Note: You may store your cloning vector after restriction enzyme digestion at – 20 °C for future use.

5. Ligation with Annealed sgRNA

In a 1.5 mL tube, mix the following:
- 9 µL of digested plasmid
- 9 µL of annealed sgRNA oligo
- 2 µL of 10X ligation buffer
- 1 µL of T4 DNA ligase
Mix gently by pipetting or tapping (do not vortex) and spin briefly.
Incubate at 16°C for 2–6 hours.

Note: The formulation mentioned above works fine in our lab, however, you may need to adjust the vector insert ratio, especially if the quality of your oligos is not good. If you face difficulties in ligation then use different ratios of vector and insert such as 1:1, 1:2, 1:3 etc.

6. Transformation into Competent Bacteria

Prepare an ice-water bath (~4 °C).
Thaw a vial of ~100 µL competent E. coli on ice.
Add ~50 µL of competent cells to the ligation reaction.
- Return the remaining competent cells immediately to –80 °C.
Mix gently by tapping the tube (do not vortex).
Incubate on ice for 10–30 minutes.
Heat shock the cells for exactly 2 minutes at 37 °C using a pre-warmed heat block.
Return the tubes immediately to ice.
Add 400 µL of room-temperature LB medium.
Plate 50 µL, 150 µL, and 250 µL on LB agar plates containing the appropriate antibiotic (e.g., Ampicillin).
Incubate overnight at 37 °C.

Note: Antibiotic selection must correspond to the resistance marker of your plasmid vector.

7. Colony Picking and Mini-Culture

Pick 12 colonies into 5 mL of LB medium containing the appropriate antibiotic.
Incubate overnight at 37 °C with shaking at 180 rpm.
Isolate plasmids from at least 6 cultures.
Perform restriction digestion to verify insert integration.

Note: Use appropriate restriction enzymes based on the plasmid map. Ideally, two different enzymes should be used unless the plasmid contains two distinct recognition sites for the same enzyme.

8. Insert Confirmation by Restriction Digestion

Prepare the digestion reaction as follows:
- 1 µg plasmid DNA
- 2 µL of the appropriate buffer
- 1 µL of restriction enzyme #1
- 1 µL of restriction enzyme #2
- Add MilliQ water to reach a total volume of 16 µL
Incubate at 37 °C for 1 hour.
Add 2 µL of 6X loading dye.
Run the sample on a 1% agarose gel.
Use a 1000 bp DNA ladder for size reference.
Capture gel images using a documentation system.

9. Sequencing of Positive Clones

Select two positive clones for Sanger sequencing to confirm the presence and integrity of the sgRNA insert.
Use an appropriate forward primer for sequencing.
- Confirm if the sequencing company provides primers.
- Refer to the plasmid information (e.g., Addgene catalog) to obtain the correct primer sequence.

Note: Sequencing is essential to confirm correct integration and to rule out unwanted mutations.

10. Optional: Large-Scale Culture and Plasmid Maxi-Prep