3.4 Genome Editing with CRISPR/Cas9

Design a sgRNA for the locus of interest using designing tools (such as http://crispr.mit.edu/, see Note 23 ) [12].

Order two oligos representing the sgRNA with a design as follows: top oligo—CACC(G)[20 N of sgRNA], bottom oligo—AAAC20 N reverse complement of sgRNA [10] ( see Note 24 ).

Anneal the oligos in a thermocycler using 2µL, 2µL, 2µL, T4 Polynucleotide Kinase, and 13µL and program the thermocycler with 0h 30m 0s at 37°C, 0h 5m 0s at 95°C, a ramp down to 25°C (ramp rate of 5 °C/min) and hold at 4°C. The oligo hybrid can directly be used for cloning into the linearized PX459 or PX458 vector.

Cut and dephosphorylate the vector using 2µL with 3µg and fill up to 50 μl reaction volume with ddH₂O (minimum of 0.5 μl BbsI for 5 μg of vector).

Incubate for 1h 0m 0s at 37°C and optionally heat-inactivate the digestion for 0h 15m 0s at 65°C.

Gel-purify the vector by running on an agarose gel and extracting the band using a gel extraction kit, such as QIAquick ^® Gel Extraction Kit (Qiagen), according to the manufacturer’s instructions. The vector can be stored at -20°C for several months.

Ligate sgRNA insert and vector in a 10 μl reaction with 100ng, 2µL, 5µL, and ddH₂O. Incubate the mixture for 0h 30m 0s at 16°C.

Transform 5µL into chemically competent bacteria, such as Stbl3. The rest of the reaction can be stored for up to 2 weeks.

Thaw the bacteria for ~0h 10m 0s On ice, add the ligation reaction, stir carefully and incubate On ice for 0h 5m 0s–0h 10m 0s. Perform a heat shock for 0h 0m 45s at 42°C transfer the sample back to ice for 0h 2m 0s–0h 5m 0s. Add 300µL and shake the bacteria at 300rpm.

Plate the bacteria on LB-plates with 100μg/mL and incubate 1h 0m 0s at 37°C and proceed with the plasmid preparation using a Miniprep kit such as the QIAprep® Spin Miniprep Kit (Qiagen) according to the manufacturer’s guidelines.

Check the construct for correctness using the respective sequencing primer and expand the DNA to a high concentration stock.

Optionally: Test the cutting efficiency with a T7 endonuclease assay [13] ( see Note 25 ).

If homologous recombination of a disease correction or knockin of a reporter is the aim of the gene editing, a donor construct has to be provided in addition to the Cas9-sgRNA vector(s). The tag or the gene for the knockin should be framed by regions of the locus in approximately the same length as the part that is transferred into the gene (such as ~1000 bp for incorporation of an antibiotic resistance with promoter) and provided as a linearized DNA fragment. Alternatively, 5–25 bp homology can be used with microhomologymediated end-joining [14].

Electroporate 10µg encoding for the correct sgRNA and optionally ~5µg into iPSCs ( see materials and steps in protocol 3.2). Seed the cells into two wells of a 6-well plate.

Using the PX459 vector, it is advisable to use 0.5μg/mL–0.8μg/mL for 12h 0m 0s–16h 0m 0s for the next night after electroporation in combination with ROCKi.

Keep ROCKi for 2 days and check if you obtained single cells that grow to small colonies. Once the colonies are visible by eye, check half of a colony for integration or knockout of the target gene by (colony) PCR with primers binding around or in the locus that you tested for specificity before (using control DNA). Transfer the other part of each colony not used for PCR to a new well (of a 48-well plate). These are the monoclonal knockout lines usable if the PCR shows the respective shifts.

Once a potentially positive clone has been detected, sequence the locus using Sanger sequencing, optimally with subcloning of the PCR product into a carrier vector (for instance using TOPO cloning).