TITLE: Generating Mutant Renal Cell Lines Using CRISPR Technologies
AUTHORS: Nuria Perretta-Tejedor, Grace Freke, Marian Seda, David. A. Long, Dagan Jenkins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles (“indels”) at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.</div></div>

[STEPS]
?. [3.1 sgRNA Design]
Find your gene of interest within the relevant species on the Ensembl genome browser (www.ensembl.org). For HEK293 search the human genome, and for IMCD3 search the mouse genome.
?. [3.1 sgRNA Design]
Select a 23–500-nucleotide (nt) genomic region that you wish to edit (see Note 3).
?. [3.1 sgRNA Design]
Open the CRISPOR design tool (crispor.tefor.net). Select the appropriate target genome and paste your sequence into the search box. Submit the search. The online tool will produce a ranked list of all possible sgRNAs within the genomic sequence (see Note 4).
?. [3.1 sgRNA Design]
Identify a suitable sgRNA sequence from the displayed list (see Note 5).
?. [3.1 sgRNA Design]
Take the 20 nt sgRNA sequence (without the PAM). If its first nucleotide is not a “G,” then add a “G” at the 5′ end. This sequence will form the foundations of oligo 1.
?. [3.1 sgRNA Design]
Find the reverse complement of the sgRNA sequence (including the extra “G” nucleotide, if added). This will form the foundations of oligo 2.
?. [3.1 sgRNA Design]
To the 5′ end of oligo 1, insert nucleotides “CACC.” Design of oligo 1 is now complete.
?. [3.1 sgRNA Design]
To the 5′ end of oligo 2, insert nucleotides “AAAC.” Design of oligo 2 is now complete.
?. [3.1 sgRNA Design]
Order oligos 1 and 2 as 5′ phosphorylated oligos.
?. [3.1 sgRNA Design]
Design a pair of PCR primers flanking the sgRNA genomic target site (see Note 6).
?. [3.2 Cloning of sgRNA into Plasmid pX330]
3.2.1 Annealing of Oligos 1 and 2
?. [3.2 Cloning of sgRNA into Plasmid pX330]
3.2.2 pX330 Linearization
?. [3.2 Cloning of sgRNA into Plasmid pX330]
3.2.3 Ligation of Annealed Oligos into pX330
?. [3.2 Cloning of sgRNA into Plasmid pX330]
3.2.4 Transformation of Competent Cells with the Ligation Mix and Isolation of Plasmid DNA
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
A different transfection reagent is used depending on the cell line (see Note 7). Choose Section 3.3.2, step 17, for transfection of HEK293 cells and Section 3.3.3, step 18, for transfection of IMCD3 cells. Regardless of transfection reagent, three transfection conditions will be used: (1) a co-transfection with pX330-sgRNA and pAcGFP1-C1 (“CRISPR” transfection); (2) a transfection with pAcGFP1-C1 (“GFP-only” transfection); (3) a mock transfection without plasmid DNA (“non-transfected” condition). The latter two conditions act as positive and negative controls of transfection, respectively.
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
3.3.1 Seeding of Cells for Transfection
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
3.3.2 Transfection of HEK293 Cells
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
3.3.3 Transfection of IMCD3 Cells
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Label a 15 mL centrifuge tube for use as a CRISPR sample collection tube. Add  to the tube, put it , and set aside.
[fresh medium]
on ice
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Wash the CRISPR and non-transfected wells of cells twice each with /well sterile DPBS (see Note 10).
1 mL
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Add/well trypsin-EDTA and incubate at  until cells dissociate.
1 mL
37 °C
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Add /well complete media and transfer each  to a 15 mL centrifuge tube.
2 mL
[cell suspension]
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Pellet cells by centrifugation at .
Centrifuge: 200 34, 5 min
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Discard the supernatant and resuspend cells in  (see Note 11).
[Opti-MEM reduced-serum media]
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Pass cells through a 70 μm cell strainer to remove clumps (see Note 12).
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Put the tubes directly .
on ice
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Proceed directly to FACS (see Note 13). Use non-transfected cells to discern single cells from cell clumps when setting up the cell sorter. Isolate GFP-positive cells from the CRISPR sample and collect these in the collection tube (prepared in Section 3.4, step 19). Run the entirety of the sample or collect at least 5 × 105 cells per sample (see Note 14). Keep collected cells .
on ice
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
In a sterile safety cabinet, take one-half of the GFP-positive CRISPR sample and seed them in a 25 cm2 tissue culture-treated flask containing . Return the cells to the incubator and continue to grow them for , passaging as and when necessary (see Note 15).
[complete medium]
?. [3.4 Group Sort by Fluorescence-Activated Cell Sorting (FACS)]
Separately pellet the remaining GFP-positive CRISPR cells and the non-transfected cells for gDNA extraction, by centrifugation at . Discard the supernatant. Proceed directly to the next step, or store the cell pellets at .
Centrifuge: 300 34, 5 min
-20 °C
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Extract genomic DNA (gDNA) with the DNeasy Blood and Tissue kit, following the manufacturer’s instructions.
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Use the extracted gDNA as template to perform a  PCR reaction using the genotyping primers (designed in Section 3.1, step 10) and Pwo Master (see Note 16).
50 µl
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Run  of the PCR in an agarose gel (adjusting the agarose percentage depending on the expected size of the PCR product) against a 1 kb DNA ladder to check whether there is a single product (see Note 17).
25 µl
?. [3.5 T7 Endonuclease I (T7EI) Assay]
In a 0.2 mL PCR tube, prepare a  reaction containing  PCR reaction,  , and .
19 µl
10 µl
[10× NEBuffer 2]
[Milli-Q H2O]
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Melt the PCR products in a thermocycler using the following conditions:  for  , ramp down to  at −2 °C/s, ramp down to  at −0.1 °C/s, and hold at .
95 °C
85 °C
25 °C
4 °C
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Add   to the melted PCR products and incubate at  for .
[T7 endonuclease I]
37 °C
?. [3.5 T7 Endonuclease I (T7EI) Assay]
Run the T7 cleavage products in an agarose gel and look for cleavage of the PCR product (see Notes 17 and Note 18; Fig. 3).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
3.6.1 Preparation of Conditioned Media
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
3.6.2 Preparation of CRISPR Cells for FACS
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
3.6.3 Expansion of Cell Colonies
?. [3.7 Genotyping of Clones]
This method describes the genotyping of a single-cell colony.
?. [3.7 Genotyping of Clones]
Extract genomic (g) DNA with the DNeasy Blood and Tissue kit, following the manufacturer’s instructions.
?. [3.7 Genotyping of Clones]
Use the extracted gDNA as template to perform a  PCR reaction with the genotyping primers (designed in Section 3.1, step 10).
50 µl
?. [3.7 Genotyping of Clones]
Run the  PCR in an agarose gel against a 1 kb DNA ladder. If there are multiple amplicons present, ensure that they are well resolved (see Notes 27 and 28).
50 µl
?. [3.7 Genotyping of Clones]
Gel-purify the PCR products.
?. [3.7 Genotyping of Clones]
Sanger sequence the extracted amplicon(s) to identify the presence and nature of mutations generated by CRISPR (see Note 29; Fig. 4).
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Resuspend each oligo (ordered in Section 3.1, step 9) to a concentration of  in Milli-Q water.
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Anneal in a thermocycler using the following cycling conditions:  for , ramp down to  at /min.
95 °C
25 °C
5 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
In a 0.2 mL PCR tube, combine  , , and .
[oligo 1 (100 μM)]
[oligo 2 (100 μM)]
[Milli-Q water]
?. [3.2 Cloning of sgRNA into Plasmid pX330]
, prepare a  reaction containing  ,  ,  , and Milli-Q water to a final volume of .
on ice
50 µl
[circular pX330 vector]
[restriction enzyme buffer (10×)]
[BbsI (5000 U/mL)]
50 µl
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Incubate for  at .
37 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Dilute annealed oligos (from Section 3.2.1, step 11) 1:200 in Milli-Q water.
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Prepare a   (from Section 3.2.2, step 12), , , , and Milli-Q water to a final volume of .
[reaction mix containing 50 ng of linear pX330]
[diluted oligos]
[10× T4 ligase buffer]
[T4 ligase enzyme]
10 µl
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Incubate for at least  at , or  at .
0 Room temperature
4 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Add  (from Section 3.2.3, step 13) to the tube of competent cells. Mix by flicking the tube 4–5 times (do not mix by pipetting) and incubate cells for .
[ligation reaction]
on ice
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Thaw E. coli JM109 competent cells  until just thawed.
on ice
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Heat shock the cells for  in a water bath at exactly ; do not shake. Immediately return the tube to ice for .
42 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Add   to the tube and incubate for  at  with shaking.
[room-temperature SOC medium]
37 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Plate   onto an LB agar Amp plate.
[transformation mix]
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Incubate plate  at .
37 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
The next day, inspect the plate for colony growth. Pick two or three colonies to check for correct insertion of the sgRNA. Inoculate a single colony into a  . Incubate cultures  at  with shaking.
[culture of LB Amp medium]
37 °C
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Isolate plasmid DNA from the cultures using a QIAprep Spin Miniprep kit, according to the manufacturer’s instructions.
?. [3.2 Cloning of sgRNA into Plasmid pX330]
Verify the sequence of each plasmid by Sanger sequencing using the pX330 sequencing primer. We will call this plasmid pX330-sgRNA.
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
The day before transfection, seed cells into three wells of a 6-well plate (see Note 8). Plate 1 × 105 to 3 × 105 cells/well in 2 mL/well complete medium. Leave  to achieve a desired confluence of 50–80%, appropriate for transfection.
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Label three sterile microcentrifuge tubes, one for each transfection condition.
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
In each tube, mix  with  and incubate for  at .
[FuGENE]
[Opti-MEM reduced-serum media]
0 Room temperature
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Add the appropriate plasmid DNA to each tube and mix. For the CRISPR transfection, add  and . For the GFP-only transfection, add . No plasmid DNA is added for the non-transfected condition. Incubate for  at .
[pX330-sgRNA]
[pAcGFP1-C1]
[pAcGFP1-C1]
0 Room temperature
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
To each well of plated cells, add  in a dropwise manner.
[transfection mixture]
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Return cells to the incubator for , prior to cell sorting (see Note 9).
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Label six sterile microcentrifuge tubes. For each transfection condition, there will be one tube for Lipofectamine and one tube for plasmid DNA.
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
In each of the three tubes, mix with  and incubate for  at .
[Lipofectamine 2000 transfection reagent ]
[Opti-MEM]
0 Room temperature
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
In the remaining three tubes, mix  with the appropriate plasmid DNA. For the CRISPR transfection, add  (obtained in step 3.2.4) and . For the GFP-only transfection, add . No plasmid DNA is added for the non-transfected condition.
[Opti-MEM]
[pX330-sgRNA]
[pAcGFP1-C1]
[pAcGFP1-C1]
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Add each plasmid mixture to a separate tube of Lipofectamine and mix well. Incubate for  at .
0 Room temperature
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
To each well of plated cells, add  in a dropwise manner.
[transfection mixture]
?. [3.3 Transfection of HEK293 or IMCD3 Cells]
Return the cells to the incubator for , prior to cell sorting (see Note 9).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
In a 50 mL centrifuge tube, make up  per 96-well plate (see Note 19), by mixing  with  medium removed from a 75 cm2 flask of cells at ~70% confluence (see Note 20).
[conditioned medium]
[fresh medium]
8 mL
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Fill a 96-well plate with 150 μL/well. Place the 96-well plate in the  incubator.
37 °C
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Sterile-filter the conditioned medium with a 0.2 μm filter (see Note 21).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Wash cells in 25 cm2 flask twice with  .
[sterile DPBS]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Wash cells in 25 cm2 flask twice with  .
[sterile DPBS]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Add   and transfer cell suspension to a 15 mL centrifuge tube.
[complete media]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Pellet cells by centrifugation at .
Centrifuge: 200 34, 5 min
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Discard the supernatant and resuspend cells in (see Note 11).
[Opti-MEM reduced-serum media]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Pass cells through a 70 μm cell strainer to remove clumps (see Note 12).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Put the tubes directly .
on ice
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Proceed directly to FACS with the cell suspension and the 96-well plate containing conditioned media. Sort a single cell into each well of the 96-well plate (see Note 22).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Spin the 96-well plate at  and immediately return them to the incubator (see Note 23).
Centrifuge: 200 34, 2 min
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
Incubate the cells for 2 weeks (), to allow colonies to establish (see Note 24).
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
After 2 weeks, check individual wells under the microscope to identify those that contain a cell colony. Monitor cell confluence in these wells over the ensuing days.
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
When a well is confluent (see Note 25), wash the cells twice in  . Then dissociate cells in   and transfer them directly into  in a well of a 12-well plate (see Note 26). Return the cells to the incubator and monitor their growth over the following days.
[sterile DPBS]
[trypsin-EDTA]
[medium]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
When a well of a 12-well plate is confluent, wash the cells twice with  .Then dissociate cells in  and transfer directly to a 25 cm2 flask containing . Return the cells to the incubator and monitor their growth over the following days.
[sterile DPBS]
[trypsin-EDTA]
[medium]
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
When a 25 cm2 flask is confluent, passage the cells into two new 25 cm2 flasks. Return these flasks to the incubator.
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
When the two 25 cm2 flasks have reached ~70% confluence, freeze down and store the cells from one of the flasks.
?. [3.6 Single-Cell Sorting of CRISPR Cells to Establish Clonal Cell Lines]
With the remaining flask, wash cells twice with , and then dissociate in . Add  and transfer the  to a 15 mL centrifuge tube. Pellet the cells for  and discard the supernatant. Proceed directly to the next step, or store the cell pellet at .
[DPBS]
[trypsin-EDTA]
[complete medium]
[cell suspension]
Centrifuge: 300 34, 5 min
-20 °C