- 1.
The given methods describe the use of the NHEJ pathway to knock out a gene by generating random indels. We have adapted these methods to knock in a specific point mutation in IMCD3 cells using the HDR pathway, by integrating methods from the literature [9, 10, 16]. The basic protocol presented in this chapter can be followed, making the following three changes:
- (a)
In Subheading 3.1, the sgRNA must be designed to target the Cas9 to cut as close to the point at which the single base change is to be introduced, ideally within 10 bp. The necessity of a PAM site being proximal to the desired mutation site is a constraint of the HDR method.
- (b)
A repair template (single-stranded oligodeoxynucleotide, ssODN) is required. This must contain the desired base change flanked by 60 nt homology arms, as well as a silent PAM mutation. Mutation of the PAM site prevents further Cas9 cleavage after mutation generation. It is important that the PAM mutation is silent to avoid introduction of an unwanted missense mutation. The ssODN should be ordered as an oligonucleotide and resuspended in Milli-Q water to a concentration of 10 μM, prior to proceeding to Subheading 3.3.
- (c)
The CRISPR transfection of IMCD3 cells (in Subheading 3.3) must accommodate the ssODN. For the CRISPR transfection, add 1.2 μg pX330-sgRNA (obtained in step 3.2.7), 1.2 μg pAcGFP1-C1, and 1 μl ssODN (10 μM).
- 2.
We have found that HEK293 cells can be transfected with high efficiency using the FuGENE 6 reagent (Promega). In IMCD3 cells, higher transfection efficiency was achieved with the Lipofectamine 2000 transfection reagent (Thermo Fisher) than with FuGENE.
- 3.
If you are not interested in editing a specific locus, the chance of gene knockout will be improved by choosing a sgRNA target site that is in an early exon of the gene. However, it is also worth assessing whether internal translation initiation codons are present within the gene, as these could generate transcripts not affected by a frameshift mutation.
- 4.
Several online sgRNA design tools are now available. We have successfully used CRISPOR (crispor.tefor.net), Chopchop (chopchop.cbu.uib.no), and Benchling (benchling.com) to design sgRNAs. Chopchop does not require the genomic region of interest to be pasted into the search box; instead, the gene of interest can be searched for within the appropriate genome, and all possible sgRNA options spanning the entire gene are presented.
- 5.
When choosing a sgRNA it is important to consider its score (out of 100). A high score indicates a faithful sgRNA. A low score indicates a sgRNA with predicted off-target activity. For each sgRNA, off-target loci (including those with mismatches) are predicted, which are listed when the sgRNA is selected. The number of off-target sites within genes is also listed. Select a high-scoring sgRNA that does not have any predicted off-target sites in genes.
- 6.
Design primers to generate an amplicon of 200–500 bp. The expected Cas9 cut site (~3 bp upstream of the “NGG” PAM) should not lie centrally within the amplicon, or else two products of roughly equal size will be produced by the T7EI assay and it will not be possible to resolve these by gel electrophoresis. It is recommended that the target genomic region, including the primer-binding sites, is Sanger sequenced within the cell line of choice. Primers should be designed to generate an amplicon that does not contain monoallelic mutations; these will give false positives in the T7EI assay and may bias primer binding (if they fall within primer-binding sites).
- 7.
In a variation of the method presented here, two sgRNAs can be used in combination to excise a genomic region. In this case, two sgRNAs should be designed in Subheading 3.1. Each can be independently cloned into a pX330 vector, to generate two separate pX330-sgRNA plasmids, one for each sgRNA. Transfection conditions can be adapted (in Subheading 3.3), to accommodate transfection of two pX330-sgRNA plasmids with the pAcGFP1-C1 plasmid. Larger deletions can be detected by running PCR products on an agarose gel (Fig. ).
- 8.
The procedure can be adjusted for other culture vessel sizes; scale reagents as necessary.
- 9.
Confirm GFP expression in CRISPR and GFP-only cells by fluorescence microscopy 18–24 h post-transfection. The GFP-only cells are used purely as a control for transfection, and can be discarded prior to the group sort.
- 10.
When preparing cells for FACS, take extra care not to cross-contaminate the different samples. The GFP-only cells do not need to be prepared for FACS (see
Note
9).
- 11.
It is important to use serum-free medium so as not to clog the FACS apparatus with sera.
- 12.
Cell straining is optional, although it is advised, particularly for IMCD3 cells. This will reduce cell clumps.
- 13.
We use a MoFlo XDP (Beckman Coulter) equipped with three air-cooled lasers: a 150 mW 488 nm argon laser, a 100 mW 643 nm red laser, and a 100 mW 355 nm UV laser. A 100 nm nozzle tip is used with 30 PSI sheath pressure. eGFP signal is collected in FL1 channel through a 530/40 band-pass filter and FL3 channel with a 613/20 band-pass filter is used to separate true GFP-positive cells from autofluorescence background. A light scatter gate drawn in the FSC versus SSC plot is used to exclude debris and clumps and include viable cells. Cells in this gate are displayed in a SSC versus SSC-W to further target single cells. Single and viable cells are then analyzed in a FL3 versus FL1 plot and a final gate is drawn on green cells for cell sorting.
- 14.
It is possible to complete the protocol if less than 5 × 105 cells are collected. In this case, prioritize cells for seeding into a culture vessel (Subheading 3.4, step 10); use a vessel of a size appropriate to the number of cells being seeded. If too few cells remain for gDNA extraction (Subheading 3.4, step 11, to Subheading 3.5, step 1), split some of the reseeded cells for gDNA extraction once they have begun to proliferate.
- 15.
We have observed that single-cell sorting cells 2–3 days post-transfection tends to give rise to heterogeneous populations of cells, rather than clonal colonies (they possess more than two alleles when genotyped). We hypothesize that this is due to continued expression of the sgRNA and Cas9 in daughter cells, after division of the single-cell sorted cell. We passage cells for 2 weeks, to await loss of pX330 plasmid from the cells.
- 16.
Optimize PCR conditions according to your needs. Pwo polymerase may be substituted for another polymerase of your choice. Ensure that the polymerase is of high fidelity and/or possesses proofreading ability (3′ to 5′ exonuclease activity); errors during PCR amplification will give false positives in the T7EI assay.
- 17.
Use non-transfected cells as a control; these should behave as WT to give a single PCR product which is not cut by the T7EI assay. Multiple PCR products may be resolved from the CRISPR sample if it contains alleles with large indels. If there is a significant size difference between these PCR products, the T7EI assay may produce more than three bands on the gel.
- 18.
sgRNA efficiency can be determined from the intensity of the products resulting from T7EI assay of the CRISPR sample: compare the intensity of the uncut product to that of the cleavage products; the brighter the cleavage products (relative to the uncut product), the more efficient the sgRNA. If T7EI assay of the CRISPR sample does not result in cleavage products, sgRNA activity may be inefficient, and design of a new sgRNA should be considered.
- 19.
Use as many 96-well plates as needed. We typically use 2–3 plates to maximize the number of colonies obtained.
- 20.
It is necessary to passage some cells (of the same line) 24–48 h before preparing transfected cells for FACS, in order to formulate conditioned medium. Conditioned medium is used to improve the likelihood of cell survival after the single-cell sort. It comprises fresh medium (containing necessary nutrients) and medium taken from cells at ~70% confluence. The latter contains signaling components secreted by healthy cells in exponential growth phase, to encourage the sorted single cells to grow.
- 21.
Conditioned medium must be sterile-filtered to remove contaminating cells, which may derive from media taken from cells at ~70% confluence. Contaminating cells will obstruct the establishment of clonal cell lines.
- 22.
The majority of cells will no longer be GFP positive. Isolate any single cells, regardless of GFP expression.
- 23.
Centrifugation of the 96-well plate improves chances of cell adhesion, by moving cells to the bottom of the plate. It is important to get the single-cell sorted cells into the incubator as quickly as possible after FACS, to maximize viability.
- 24.
Changing medium is not necessary; this risks loss of cells from the wells.
- 25.
It may be necessary to passage the cells before the entire well is confluent, if cells become dense but do not spread across the well, and media become orange/yellow in color.
- 26.
Confirm cell detachment under the microscope prior to transferring cells to the new culture vessel. Transfer the full volume of cell suspension to the 12-well plate without pelleting cells. Then check the (empty) well of the 96-well plate under the microscope again, to ensure that all cells have been transferred. Loss of cells during passage will result in reduced cell density in the 12-well plate, which may decrease cell viability.
- 27.
Presence of more than two PCR products indicates that the colony is a clone with aneuploidy, or that it is not clonal (it is a heterogeneous population).
- 28.
It is possible to perform the T7EI assay to genotype clones. However, note that T7EI will not cleave PCR products derived from homozygous mutants. T7EI will only give a positive result (cleavage) in PCR products derived from heterozygotes or compound heterozygotes.
- 29.
If Sanger sequencing produces two overlapping traces, the clone is a heterozygote or a compound heterozygote. Cloning of PCR products in pGEM-T Easy vector systems (Promega) can be used to isolate allelic sequences. If more than two overlapping traces are seen, the colony is a clone with aneuploidy, or it is not clonal (it is a heterogeneous population).