TITLE: Basic Protocol 2: Tagging a gene of interest with AID
AUTHORS: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The next step in developing the ARF-AID system is to tag the gene of interest with AID. The ARF-AID system requires full-length AID (Figure 1) because the characterized interaction domains of AID with ARF are domains III and IV. Domains I and II are involved in the interaction with TIR1. The mini-AID lacks domains III and IV and will not interact with ARF to stabilize the protein in the absence of auxin (Sathyan et al., 2019). Note that the antibiotic selection marker (HygroR) is co-transcribed with AID and the protein products are separated during translation. Therefore, the resistance marker will be expressed at levels comparable to the target protein.</div></div>

[STEPS]
?. [Cloning the guide RNA into pSpCas9(BB)-2A-GFP (pX458) – Digestion of the vector with BbsI]
Digest  of pX458 with BbsI  at BbsI reaction mix:NEB buffer 2.1         5.0 µl BbsI enzyme            1.0 µl 100X BSA                 0.5 µl pX458 plasmid        3.0 µg Water                        to 50 µl
3 µg
37 °C
The overnight digestion with BbsI significantly reduces negative colonies.
?. [Gel purification of the digested plasmid]
Add  loading dye to the mixture and load onto  agarose gel. Use the same amount of the undigested pX458 vector as a negative control.
8 µl
?. [Gel purification of the digested plasmid]
Use a transilluminator to excise the digested band using a clean scalpel and use the QIAquick gel extraction kit (Qiagen, 28704) to isolate the digested plasmid.
The use of the blue light dark reader transilluminator (Clare Chemical Research, USA) may reduce DNA damage during gel excision. The digested plasmid runs between supercoiled and relaxed DNA in the undigested control sample.
?. [DNA purification]
Add three volumes of buffer QG to one volume of gel; consider  of gel equivalent to . For example, add  of QG buffer for  of gel.
The method described here is adapted from the kit manual with minor changes (QIAquick gel extraction kit, Qiagen, 28704).
1 mg
1 µl
300 µl
100 mg
?. [DNA purification]
Incubate at  for . Mix by vortexing every  to dissolve the gel. Once the gel has completely dissolved, add  of  sodium acetate, , irrespective of the color of the mixture, and briefly mix by vortexing.
50 °C
10 µl
The addition of sodium acetate significantly increases the final yield of the DNA.
?. [DNA purification]
Add one gel-volume of isopropanol and vortex briefly.
?. [DNA purification]
Transfer the mixture into the QIAquick column and centrifuge at  for .
Centrifuge: 17000 34
If there is more dissolved gel mixture left, add to the same column and repeat step 7.
?. [DNA purification]
Remove the flow-through and place the column back to the collection tube. Add  QG buffer to the column and centrifuge at  for .
500 µl
Centrifuge: 17000 34
?. [DNA purification]
Remove the flow-through and place the column back to the collection tube. To wash the column, add  of PE buffer and centrifuge at  for .
750 µl
Centrifuge: 17000 34
?. [DNA purification]
Discard flow-through and replace the column into the same tube and centrifuge again at  for .
Centrifuge: 17000 34
?. [DNA purification]
Place the column into a new clean 1.5 ml Eppendorf tube and add  DNase and RNase free water. Incubate for  and centrifuge at  for .
50 µl
Centrifuge: 9000 34
Centrifuging at 9000 x g reduces the chance of breaking off the Eppendorf tube’s lid.
?. [DNA purification]
Quantify DNA using a NanoDrop and store at
-20 °C
?. [Ligation of guide sequence into pX458 plasmid]
Make  solution of the forward and reverse strands of the sgRNA guide sequence in nuclease free water.
?. [Ligation of guide sequence into pX458 plasmid]
Phosphorylate and anneal the forward and reverse oligonucleotides of the sgRNA guide sequence in one reaction.Phosphorylation and annealing reaction mix: Forward (100 µM)         1 µl Reverse (100 µM)          1 µl T4 DNA Ligase buffer   1 µl T4 PNK                            1 µl Water                               6 µlIncubate in a thermocycler Ramp down to  at /min rate.
37 °C
95 °C
4 °C
4 °C
?. [Ligation of guide sequence into pX458 plasmid]
Dilute the annealed sgRNA guide sequence pair 20x by adding  of nuclease-free water. We use one microliter of this  dsDNA product for ligation.DNA ligation mix: BbsI digested pX458         50 ng annealed sgRNA                1 µl T4 ligase Buffer                  1 µl T4 DNA Ligase                   1 µl Water                                   to 10 µl
190 µl
?. [Ligation of guide sequence into pX458 plasmid]
Incubate ligation mix for  at  or . Include no insert control to see the rate of negative colonies.
0 Room temperature
16 °C
Typically, we incubate 2 hours at room temperature and start the transformation. Keep the rest of the ligation mix at 16oC overnight. If the first transformation does not result in colonies, then retransform the ligated product the next day.
?. [Ligation of guide sequence into pX458 plasmid]
Add  of the ligated product to  chemically competent E. coli. We used Max efficiency DH5a competent cells from Invitrogen for transformation.
2 µl
20 µl
?. [Ligation of guide sequence into pX458 plasmid]
Incubate  for  and then heat shock for  at .
on ice
42 °C
?. [Ligation of guide sequence into pX458 plasmid]
Put back  for .
on ice
?. [Ligation of guide sequence into pX458 plasmid]
Add  LB or SOC media and incubate at  for
250 µl
37 °C
?. [Ligation of guide sequence into pX458 plasmid]
Plate the cells () on a carbenicillin plate (stable version of ampicillin) and incubate at .
272 µl
37 °C
?. [Ligation of guide sequence into pX458 plasmid]
Pick three colonies from each plate and inoculate in LB containing carbenicillin and incubate  at .
37 °C
?. [Ligation of guide sequence into pX458 plasmid]
Pellet the bacterial culture by centrifugation at  for  and follow the steps in the QIAprep spin miniprep kit. Elute DNA in  nuclease free water.
Centrifuge: 17000 34
50 µl
?. [Ligation of guide sequence into pX458 plasmid]
Confirm the sgRNA insertion by sequencing using the LKO.1 5' primer or the U6 promoter primer.
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Plate 30% confluent HEK293T cells in 6 well plate (start with 2 x 105 cells).
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Transfect  of sgRNA plasmid with lipofectamine 3000 reagents as described in steps 5 – 9 from basic protocol 1.
1 µg
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Twenty-four hours after transfection, replace media with  new media.
2 ml
GFP in the pX458 plasmid allows easy assessment of the transfection efficiency. Observe the transfected cells under a fluorescent microscope using a green filter. Higher transfection rates make it easier to assess the efficacy of the sgRNA using surveyor assay
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Seventy-two hours after transfection, remove  of media and collect the cells in the remaining  of media by pipetting up and down and transfer into a 1.5 ml tube.
1 ml
1 ml
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Centrifuge cells at  for  using a fixed angle rotor table top centrifuge and remove media. Cells can be stored at  or directly begin genomic DNA isolation.
Centrifuge: 6000 34
-20 °C
?. [Testing sgRNA using surveyor assay –  Transfection of the sgRNA construct]
Proceed with genomic DNA isolation, as described in steps 25 – 35 from basic protocol 1.
?. [Genomic DNA PCR – Primer design]
Make a PCR master mix by adding all the components except genomic DNA and gene specific primers adjusted for the total number of reactions.
To amplify the region targeted by the sgRNA, design a set of forward and reverse primers. The forward primer should be ~250 bp upstream from the sgRNA cutting site and the reverse primer should be ~250 bp downstream. To facilitate mutation detection, we recommend an amplicon size of 500bp to 1Kb with an sgRNA cut site close to the center of the amplicon. There are several tools to pick appropriate primer sets, such as from Primer3, IDT or Benchling (Untergasser et al., 2012; Benchling, 2019) https://www.idtdna.com/pages/tools/primerquest). The double stranded DNA breaks generated by CRISPR-Cas9 are typically repaired by error prone non-homologous repair, which results in several types of mutations. The mutated PCR products, when annealed with the wild type or other mutated PCR products, generate a mismatch proximal to the cut site. The surveyor enzymes recognize this mismatch and cleave the heteroduplex at the site of mismatch (Figure 6). The assay produces three fragments, one undigested and two digested fragments. Depending on the site of mismatch, the digested fragments run either as one or two bands (Figure 6B&C). If the sgRNA cutting site is at the center of the PCR product, it generates two unresolvable fragments, whereas unequal fragments run as distinct bands. There is no need to mix wild type PCR products with the PCR products of the sgRNA transfected cells for the assay because many different repair products will form and other sites in the population will remain unmodified. One set of primers is required for each terminus of the gene to test sgRNAs that target these sites.We used Platinum Taq DNA polymerase or Platinum Taq DNA Polymerase High Fidelity for genomic PCR. For each primer set, it is necessary to determine the annealing temperature empirically. We find that an annealing temperature within 3 degrees of the lowest melting temperature (Tm) works well. Additionally, adding denaturants such as DMSO or Betaine may help to amplify GC rich genomic regions.
?. [Genomic DNA PCR – Primer design]
Aliquot  of the master mix into 0.2 ml PCR tubes and add  of  genomic DNA into each reaction mix. The parental HEK293T DNA serves as a negative control.
18 µl
5 µl
?. [Genomic DNA PCR – Primer design]
Add  each of forward and reverse primers of the corresponding gene targeted.
1 µl
?. [Genomic DNA PCR – Primer design]
PCR mix:Genomic DNA                                                                  5.0 µl Gene Primer F                                                                  1.0 µlGene Primer R                                                                  1.0 µl 10X HF buffer                                                                   2.5 µl DMSO (100%)                                                                   0.5 µl 10 mM DNTP                                                                     1.0 µl MgSO4                                                                               1.0 µl Platinum Taq DNA Polymerase High Fidelity             0.5 µl Water                                                                                12.5 µlRun PCR as follows: Initial denaturation 30 cycles of  ___°C.  (Annealing temperature changes based on the primers used) Final extension  hold ∞
Annealing and extension of the PCR steps depends on the primers and Taq DNA polymerase used.
95 °C
95 °C
68 °C
68 °C
4 °C
?. [Genomic DNA PCR – Primer design]
Check PCR amplification by running  of the products on an agarose gel. Add  6x DNA sample buffer to  of PCR product and load onto a  agarose gel.
5 µl
2 µl
5 µl
Keep the remainder of the PCR product for the surveyor assay. This step makes sure the PCR worked before starting the surveyor assay.
?. [Genomic DNA PCR – Primer design]
Run the samples at constant 90V and visualize the bands using a UV transilluminator (follow steps 38 – 42 from basic protocol 1). If there is only one bright PCR product, proceed with the surveyor assay.
?. [Surveyor assay]
Denature the PCR products from  at  for  and then allow to renature stepwise using the following program: hold ∞ A ramp down rate of /sec is recommended.
This step involves denaturation followed by annealing of the PCR products to form heteroduplexes, followed by a surveyor nuclease reaction. The denaturation and renaturation step is essential because the final cycle of PCR generates homoduplexes that are not recognized by the surveyor nuclease.
95 °C
95 °C
85 °C
75 °C
65 °C
55 °C
45 °C
35 °C
25 °C
4 °C
0.3 °C
?. [Surveyor assay]
Surveyor nuclease reaction mix: Reannealed PCR product         20.0 µl Surveyor Nuclease S                 1.0 µl Surveyor Enhancer S                 1.0 µl 0.15 M MgCl2 Solution             2.0 µl
?. [Surveyor assay]
Mix by pipetting and incubate at  for  in a thermocycler.
42 °C
?. [Surveyor assay]
Stop the reaction by adding  of Stop Solution and mix.
2.4 µl
Either directly electrophoresis the sample on a 2% agarose gel after mixing with loading dye or store at -20°C for future use.
?. [Surveyor assay]
Run the samples at constant 90V and visualize the bands using a UV transilluminator. Follow steps 38 – 42 from basic protocol 1 for running and visualizing the samples. The ratio of the undigested band with that of the digested band gives an estimate of relative sgRNA efficiency.
As an example, we designed two sgRNA that target the 3' end of FOXM1 coding regions (Figure 6B&C).
?. [PCR amplification of Homology Directed Repair construct]
Synthesize the designed primers from Integrated DNA Technologies (IDT), adding phosphorothioate as a modification to the primer in the details.
?. [PCR amplification of Homology Directed Repair construct]
Amplify the HDR template using the primers and Platinum Taq DNA Polymerase High Fidelity. We perform several 50 µl reactions (typically 4 to 8), and gel purify the PCR products.PCR mix: Plasmid DNA                                                             50.0 ng 10 µM Primer F                                                         1.0 µl 10 µM Primer R                                                         1.0 µl 10XHigh Fidelity buffer                                            5.0 µl DMSO                                                                         0.5 µl 10 mM DNTP                                                             2.0 µl MgSO4                                                                       2.0 µl Platinum Taq DNA Polymerase High Fidelity      0.5 µl Water                                                                         to 50 µlUse PCR condition as follows: Initial denaturation 30 cycles of Final extension  hold ∞
95 °C
95 °C
60 °C
68 °C
68 °C
4 °C
Removing any remaining primer from the PCR products is important as primers may interfere with homologous recombination. The primers are very long, so conventional PCR clean-up kits will not remove primers efficiently and reduce the HDR efficiency by binding to the cut site. Always gel purify the PCR products.
?. [Agarose gel purification]
Run the PCR products on an agarose gel and cut out the repair construct band.
?. [Agarose gel purification]
Combine all the gel slices into one tube and purify the DNA using the Qiagen gel purification kit similar to steps 2 – 12.
?. [Agarose gel purification]
Elute DNA using  to  nuclease-free water and quantify using a NanoDrop and store at . DNA is stable in  for several months.
50 µl
100 µl
-20 °C
-20 °C
?. [Transfection]
Grow the ARF-AID progenitor cells and split into six-well plates to ~30% confluency.
?. [Transfection]
Cotransfect  of gene-specific sgRNA plasmid and  of double-stranded homology repair template PCR product to the cells as described in steps 1 – 9 of basic protocol 1. Use  –  Lipofectamine reagent for transfection. The parental pX458 cotransfected with HDR PCR product is used as a transfection control and we recommend keeping a untransfected control cell well.
1 µg
400 ng
5 µl
7.5 µl
Transfect multiple (at least 4 wells) wells to get enough colonies.
?. [Transfection]
Replace with new media 24 hours after transfection.
?. [Transfection]
After forty-eight hours, expand each well into a 10 cm plate.
Expanding cells into 10 cm plates spreads cells so they form isolated colonies.
?. [Transfection]
Seventy-two hours after transfection, add  final concentration of hygromycin B to the cells and keep in the selection media until all cells in the untransfected control condition die. Antibiotic selection concentration varies between cell types. We recommend plotting a titration vs. cell viability curve to determine the lowest concentration at which nearly all cells die within 7 – 12 days.
If the cell number is low after 72 hours, start hygromycin B selection on the 5th day of transfection. This increases the chance of getting positive colonies. The addition of conditioned media (8:2 ratio of new media with filtered cultured media from the same type of cell) increases the survival of the clones.
?. [Transfection]
After all the cells in the control plate are dead, replace the media with regular conditioned media in all the transfected cells and control plates.
?. [Transfection]
Colonies will appear in the plates after two or three weeks.
?. [Transfection]
Pick colonies similar to the steps 15 – 22 (from basic protocol 1) and screen for tagged clones by Western blotting.
We use gene specific antibodies for detecting tagged proteins. The successful tagging results in an increase in protein size of 24 KD. Heterozygous integration will contain bands reflecting both the native and tagged proteins. The non-homologous end repair may produce read through into the 3' UTR or introduce stop codons before normal stop codon of the gene. This is observed as an unexpected size shift that is not ~24KDa. Confirm the integration and reading frame of the integrated AIDP2A-HygR or HygR-P2A-AID by PCR and sequencing.
?. [Transfection]
Select colonies from the Western blotting experiment and perform genomic DNA PCR using the same set of primers used for testing sgRNA efficiency according to steps 31 – 34, with the following change: PCR extension time should be increased to  to amplify the insert.
?. [Transfection]
Electrophorese whole PCR product on  agarose gel, excise the band using a clean scalpel and use the Qiagen gel purification kit to isolate genomic DNA (follow steps 2 – 12). Sequence the purified PCR product with the forward primer to confirm the reading frame.
Successful integration of the AID-P2A-HygR or HygR-P2A-AID results in an addition of approximately 1785 bp to the PCR product. Heterozygous clones will have two bands, one with a length of genomic region between forward and reverse primer and the second with a length of genomic region between primers plus the 1785 bp. The homozygous integration will result in one band with a length of genomic region between primers plus the 1785 bp. Sequence the integrated DNA using the same forward primer, which will confirm successful integration.