CRISPR-SCReT (CRISPR-Stop Codon Read Through) method to control Cas9 expression for gene editing.

Abstract

CRISPR/Cas9 has paved the way for the development of therapies that correct genetic mutations. However, constitutive expression of the Cas9 gene can increase off-target mutations and induce an immune response against the Cas9 protein. To limit the time during which the Cas9 nuclease is expressed, we proposed a simple drug inducible system. The approach consists of introducing a premature termination codon (PTC) in the Cas9 gene and subsequently treating with an aminoglycoside drug, which allows readthrough of the complete protein. To validate that system, HEK293T cells were co-transfected with a PX458 plasmid, which was mutated to introduce a PTC in the SpCas9 gene and two sgRNAs targeting the DMD gene (exons 50 and 54). Cells were treated with different doses of geneticin (G418) for 48 h. Western blot confirmed that the Cas9 protein expression, which was shut down by the PTC mutation, can be induced by the drug. The hybrid exon 50-54 formed by the deletion of part of the DMD gene was detected by PCR only in the cells treated with G418. The approach was also used successfully with CjCas9 to edit the FXN gene. Our results show that it is possible to control SpCas9 and CjCas9 expression by CRISPR-SCReT (CRISPR-Stop Codon Read Through) method.