Efficient in vitro and in vivo self-repression of SpCas9 gene using a molecular Hara-Kiri method.

Abstract The CRISPR/Cas9 system is currently a major revolution in the field of biology. Because of its simplicity compared to other endonucleases, this system is being experimented in diverse fields. However, a major disadvantage is the toxicity linked to sustained Cas9 expression. In the present study, we present an approach to effectively suppress the expression of the Streptococcus pyogenes Cas9 (SpCas9) gene. This approach that we call the molecular Hara-Kiri method, involves two sgRNAs targeting two sequences in the SpCas9 gene. The SpCas9 enzyme binds to the Protospacer Adjacent Motifs following the two sequences targeted by the sgRNAs and induces two Double Strand Breaks (DSBs) in its own gene (Hara-Kiri). The sequence located between the DSBs is then deleted. Most of the time, the SpCas9 gene is repaired by Non-Homologous End Joining without INDELs. By adequately selecting the targeted sequences, the junction of the SpCas9 gene residues generates a TAA type stop codon within this truncated gene to effectively suppress its expression. This results in dramatic decrease of the SpCas9 protein in vitro and in vivo.