1. Pre-sgRNA plasmids cloning
I) Digesting pre-sgRNA plasmid with EcoRI-HF and BamHI-HF with the following reaction:
500 ng PB-CAGGs-zeocin plasmid
1 μl CutSmart buffer
0.5 μl EcoRI-HF
0.5 μl BamHI-HF
Add H2O up to 10 μl.
Incubate 2 hours at 37° C.
After the digest, gel purify the linearized plasmid.
II) Clone pre-sgRNA
Pre-sgRNA contains a sgRNA sequence flanked by two miRNA complementary binding sites.
Example pre-sgRNA sequence:
294T-sgRNA(TRE)-294T:
(ACACACAAAAGGGAAGCACTTT)tacgttctctatcactgataGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC(ACACACAAAAGGGAAGCACTTT)
Bases in () indicate the miRNA binding sites which are reverse and complementary with mature miRNA sequence (miR-294-3p). Nucleotides in the middle outside of () indicate the sgRNA(F+E) sequence23, while the first 20 nucleotides of sgRNA (lower case) are crRNA sequences which target to TRE promoter. We recommend using the “Guide Design Resources”: https://zlab.bio/guide-design-resources) to facilitate target selection.
a) Order the following oligos:
Oligo1 (Forward) for the example pre-sgRNA sequence:
AGAATTCACACACAAAAGGGAAGCACTTTTACGTTCTCTATCACTGA
Oligo2 (Reverse) for the example pre-sgRNA sequence:
AGGATCCAAAGTGCTTCCCTTTTGTGTGTGCACCGACTCGGTGCCAC
b) Carry out PCR reactions on a thermal cycler as follows:
95° C for 2 minutes; 35 cycles of 95° C for 30s, 60° C for 30s, 72° C for 30s; 72° C for 5 minutes; 4° C forever.
c) Run raw PCR product on 1.5% agarose gel electrophoresis, separate 164 bp amplicon and purify with HiPure Gel Pure DNA Mini Kit (Magen).
d) Digest purified PCR product with BamHI-HF and EcoRI-HF via the following reaction at 37° C for 2 hours:
26 μl Purified PCR product
3 μl Cutsmart buffer
0.5 μl EcoRI-HF
0.5 μl BamHI-HF
total 30 μl.
e) After digestion, purify with HiPure Gel Pure DNA Mini Kit (Magen).
f) Ligate pre-sgRNAs into the vector with reactions as follows:
1 μl Linearized PB-CAGGS-zeocin vector
7 μl Pre-sgRNAs
1 μl T4 DNA ligation buffer
1 μl T4 DNA ligase
g) Incubate at room temperature for 1 hour.
h) Transform into Trans-T1 Phage Resist Chemically Compotent Cell (Transgen) according to the manufacturer’s protocol.
i) Use forward primer inside CAGGS promoter and reverse oligonucleotides as a reverse primer for PCR screening. Choice colonies which have a single band around == ~== 250 bp. Confirm the sequence of pre-sgRNA by Sanger Sequencing.
j) Extract plasmids using AxyPrep Plasmid Miniprep Kit (Axygen). Now plasmids are ready to use.
2. Generate miR-294-sensing-CRISPR-on mouse embryonic stem cells.
Day1
Plate 50,000 mouse embryonic stem cells in 24-well plates, using standard growth medium.
Day2
Replace with 500 μl of growth medium.
Prepare A mix with 200 ng dCas9-VPR, 200 ng 294T-sgRNA-294T, 100 ng TRE3G-RFP, 100 ng pBase and 1 μl P3000 Reagent in 50 μl OPTI-MEM. Add 1.25 μl Lipo3000 in 50 μl OPTI-MEM to get B mix. Vortex each mix well. Combine A and B mix, vortex thoroughly and incubate 5 minutes at room temperature. Add the solution to the cells carefully.
Day3
Replace with 500 μl growth medium.
Day4-7
Cells are treated with 10 μg/ml Blasticidin S (Gibco), 150 μg/ml Hygromycin (Roche), 100 μg/ml Zeocin (Invitrogen).
Day8
Plate 500 cells on feeder for colony picking.
3. MICR-ON and MICR-i
For endogenous gene activation
Day1
Plate 50,000 HEK293T cells per well in poly-D-lysine-coated 48-well plate.
Day2
18 hours later replace with 500 μl of growth medium. Mix 20 nM final concentration of miR-122 or NC in 25 μl OPTI-MEM \(amounts and volumes for a single well). Mix 0.5 μl DharmaFect 1 Transfection Reagent in another 25 μl of OPTI-MEM. Vortex to mix two solutions separately, incubate 5 minutes at room temperature.
Combine the two solutions, mix gently and incubate 20 minutes at room temperature.
Add the solution to the cells carefully.
After 6 hours, replace medium with standard growth medium. Prepare A mix with 125 ng dCas9-VPR plus 125 ng pre-sgRNA plasmids or empty control plasmids and 0.5 μl P3000 Reagent in 25 μl OPTI-MEM. Add 0. 6 μl Lipo3000 in 25 μl OPTI-MEM to get B mix. Vortex each mix well. Combine A and B mix, vortex and incubate 5 minutes at room temperature. Add the solution to the cells carefully.
Day4
48 hours after transfection, cells were harvested with Trizol for extracting total RNA for mRNA qRT-PCR \(mRNA Universal SYBR qPCR Master Mix, Vazyme).
For endogenous gene repression, use dCas9-KRAB instead of dCas9-VPR.
4. MICR-BE
Day1
Plate 50,000 Hela cells per well in 24-well plate.
Day2
18 hours later replace with 500 μl of growth medium.Mix 50 nM final conctration of miR-294 or NC in 50 μl OPTI-MEM \(amounts and volumes for a single well). Mix 1 μl DharmaFect 1 Transfection Reagent in another 50 μl of OPTI-MEM. Vortex to mix two solutions separately, incubate 5 minutes at room temperature.
Combine the two solutions, mix gently and incubate 20 minutes at room temperature.
Add the solution to the cells carefully.
After 6 hours replace medium with standard growth medium. Prepare A mix with 250 ng dCas9-Apobec1-UGI (BE3) plus 250 ng pre-sgRNA plasmids or empty control plasmids and 1 μl P3000 Reagent in 50 μl OPTI-MEM. Add 1.25 μl Lipo3000 in 50 μl OPTI-MEM to get B mix. Vortex each mix well. Combine A and B mix, vortex and incubate 5 minutes at room temperature. Add the solution to the cells carefully.
Day4
48 hours after transfection, cells are treated with 500 μg/ml Hygromycin (Roche), 100 μg/ml Zeocin (Invitrogen).
Day6
After 2 days, cells are harvested for extracting genomic DNA. Genomic regions are amplified with PCR with primers designed by “Primer-BLAST (NCBI)”:https://www.ncbi.nlm.nih.gov/tools/primer-blast/. Carry out PCR amplification as follows:
95° C for 10 minutes; 35 cycles of 95° C for 30s, 60° C for 30s, 72° C for 30s; 72° C for 5 minutes; 4° C forever.
PCR products are purified using spin columns in Universal DNA Purification Kit (TIANGEN Biotech).
Anneal and digest purified PCR product with ApaI.
Assemble reaction as follows:
200 ng PCR product
2 μl 10X NEBuffer 3.1
Add H2O up to 19 μl.
Heat up the reaction to 95° C, using thermocycler for 5 minutes and let it cool down to room temperature.
Add 1 μl ApaI to the reaction, incubate 25° C for 3 hours.
Run digested product on 2% agarose gel under 120V for 30 minutes.
The intensity of PCR amplicon and cleaved bands is quantified using ImageJ. For each lane, the fraction of cleaved products is calculated by the following formula: Fc = (b+c)/(a+b+c), where a is the intensity of the undigested PCR product and b and c are intensities of each cleavage products. Sequence editing efficiency = 1-Fc1/2.