Knockout

Elimination of HIF1A

The genetic circuit designed to control chimeric antigen receptor (CAR) expression in the tumor microenvironment relies on exclusive control of hypoxia-inducible factor 1 alpha (HIF1A) by the introduced AND gate. Various strategies have been employed to obtain HIF1A deficient cells.

CRISPR/Cas9 with HDR

The CRISPR/Cas9 (Ran et al. Cell 2013) system (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) allows precise editing of genes. The Cas9 endonuclease forms a complex with guide RNA (gRNA) and introduces DNA double strand breaks at sites complementary to the gRNA. These are repaired by different endogenous repair mechanisms, dependent on the circumstances. One process called homology-directed repair (HDR) can be triggered by co-transformation with a repair template containing regions homologous to the target.

A plasmid kit with Cas9 from S. pyogenes was used to knockout HIF1A by HDR (sponsored by OriGene). This was conducted with two plasmids containing different gRNAs, both targeting the first exon of human HIF1A, close to the transcription initiation site (Fig. X). The reporter genes green fluorescent protein (GFP) and puromycin resistance were stably integrated via a repair template.

CRISPR/Cas9 induced NHEJ

The CRISPR/Cas9 system can also be used without repair template. This forces cells to repair the double strand break by non-homologous end joining (NHEJ), an error-prone process. At the cleavage site nucleotides are randomly added and removed during repair resulting in deletions, substitutions and insertions. This stochastically produces a shift of the translational frame in the gene, ablating the gene product’s function.

For this strategy gRNAs were designed using the Broad Institute’s Genetic Perturbation Platform (GPP; Doench, Fusi et al., Nature Biotechnology 2016). Off-target effects were predicted using the program CRISPR/Cas9 Target Online Predictor (CCTOP; Stemmer et al. PLOS ONE 2015). The most promising candidates were inserted into Cas9 (S. pyogenes) plasmids with either GFP or puromycin resistance as selection marker.

RNA Interference

Another technique to inhibit gene expression is RNA interference, where translation is inhibited by antisense RNA binding to target mRNA (Fire et al. Nature 1998). This cleaves the mRNA or prevents association of the translational machinery and therefore protein production. Our approach involved stable lentiviral transduction of such short hairpin RNA (shRNA) into cells to obtain continuous downregulation of HIF1A mRNA. These shRNAs rely on the endogenous processing mechanisms of cellular regulatory RNA (Figure 2).

Since the knockdown of HIF1A gene product in our project serves to allow control of this gene’s expression by a synthetic construct, it is crucial that the designed shRNA sequences are complementary to endogenous mRNA but not that of the introduced gene. Therefore, untranslated regions of this transcript were used as target for shRNA sequence design. The lentiviral transfer plasmid into which shRNA sequences were cloned contained cyan fluorescent protein (CFP) and neomycin resistance markers.

Figure 1: Schematic mechanism of RNA interference.
After translation of the siRNA gene, the precursor shRNA is processed before and after export from the nucleus. Mature siRNA associates with several proteins into the RNA-induced silencing complex (RISC) which is able to silence the desired gene via blocking translation by the ribosome or by initiating degradation of the target mRNA.

Methods

CRISPR strategies were applied by transient transfection and knockdown was lentivirally transduced followed by sorting of fluorescence positive cells. Insertion of mutations was assessed by PCR or T7E1 assay and the absence of gene product was confirmed by Western Blot.

AND Gate