Cell Engineering (Ralf Schülein)

1. Knock-outs in target genes (KOs)

At the moment, we use a vector-based system (Fig. 6) and a protein-based electroporation system (Fig. 7) for gene editing. For the vector-based strategy, we clone the sgRNAs of your target gene into the pGuide-it-zsgreen1 plasmid. The vector expresses Cas9 and can be directly used for transfection of the target cells. To preselect transfected cells, the vector expresses also GFP. A more detailed protocol of the experimental procedures leading to gene editing is outlined in the right panel of Fig. 6. Using this vector, we will usually be able to knock-out both alleles of the target gene at the same time. Even in the case of a triploid target (e.g. genes encoded on the triploid X chromosome of HEK 293 cells) a simultaneous knock-out is possible – although more clones must be analyzed in this case. For the protein-based electroporation strategy (Fig. 7), we use purified Cas9 protein. It is complexed with either in vitro transcribed and purified sgRNA or with a commercially available, synthetic full length sgRNA. Target cells are electroporated with this binary complex using the Neon electroporation system.  A more detailed protocol of the experimental procedures is outlined in Fig. 7 .

2. Knock-ins in target genes (KIs)

For KIs, we will use the electroporation protocol described above. The specific binary sgRNA/Cas9 complexes will be delivered together with a single-stranded DNA (ssDNA) repair template into cells.  In comparison to a double-stranded DNA (dsDNA) repair template, such as a plasmid or a PCR fragment, ssDNA templates are less frequently integrated into the genome at random sites and are less toxic during the electroporation procedure. For preparation of the ssDNA repair template, a PCR fragment containing the KI sequence and two homology arms (300-600 bp) will be amplified. The ssDNA will then be generated by selective enzymatic digestion of the phosphorylated strands of the PCR fragment.

3. Genotyping

A KO in both alleles of a target gene can be confirmed by immunoblotting provided that your antibody is specific (you may learn that some antibodies are not). In case that a specific antibody is not available, PCR amplification of the target gene and sequencing and its sequencing is recommended. However, sequencing alone does not tell you unambiguously whether the cell clone is homozygous or heterozygous for the indel since in the heterozygous situation, there may be a preference for one of the two alleles during PCR amplification. We thus finally prefer genotyping of the cell clones to get clear results (Fig. 8). We start with genomic DNA isolation and amplification of a segment containing the gene of interest. This DNA fragment is then incubated with by the purified Cas9/sgRNA complex which was originally used for gene editing. If no allele was modified, i.e. if both are still WT, Cas9 cleaves again and two bands could be seen on an agarose gel (Fig. 8, left lane). In a heterozygous situation, three bands will be seen instead: one large band representing the mutant and un-cleaved fragment and two bands representing the WT, cleaved fragments (central lane in Fig. 8). In case of a KO of both alleles, only one larger band will be seen representing both mutant fragments at the same size (right lane in Fig. 8).

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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