130. Gene Editing of the AAVS1 Locus with CRISPR/Cas9

One strategy for gene therapy is gene editing with engineered nucleases such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) or the CRISPR/Cas9 system. The latter uses the Cas9 endonuclease and a small guide RNA (sgRNA) to direct specific DNA double-stranded breaks at the chosen target site. Once the DNA breaks have occurred, they are repaired by the natural process of homologous recombination (HR). The introduction in the cell of a HR donor template allows replacement of a defective gene with a normal allele at its natural location. In order to improve the Cas9 nuclease activity and specificity for a target sequence, we altered the structure of the sgRNA by shortening the sequence complementary to the target site and lengthening the binding sequence of Cas9. We tested the efficacy of these modified sgRNAs on the adeno-associated virus integration site 1 (AAVS1) in human cells transduced by lentiviral vectors. We used an Integration-deficient Lentiviral vector (IDLV) carrying both cassettes of Cas9 and a modified sgRNA targeting AAVS1, together with another IDLV carrying a GFP donor cassette integrating at AAVS1 site after HR. We observed similar level of Cas9 nuclease activity with sgRNAs bearing shorter complementary sequence, compared to the non-modified sgRNAs, at the AAVS1 site. Moreover, by increasing the length of the scaffold RNA part of the sgRNA, we significantly increased the rate of homologous recombination at the AAVS1 site. The improvement of the sgRNA structure tested on the AAVS1 locus can benefit to the targeting of other genome sequences by the CRISPR/Cas9 system.