Figure 3.

Exon 13 Is Efficiently Edited in HEK293 Cells with sgRNA-1

(A) Diagram depicting the targeted region and the two specific ssODNs used as an exogenous template for repair. The 193-nt-long ssODN-2299 is shown above, highlighting the guanine deletion corresponding to the c.2299 position. Below, the 197-nt-long ssODN-2276 presents a similar composition, but with c.2276G>T change instead of the c.2299delG. The diagram also displays the silent mutation c.2292G>C placed in both ssODN designs. This variant is located in the PAM sequence to avoid recognition of the target by the sgRNA, and, therefore, to be cleaved by Cas9. Moreover, this variant creates the new recognition site for the MlsI restriction enzyme for the edition detection. (B) RFLP results of HEK293 transfection with 3 μg sgRNA-1 together with 10 pmol of the ssODN including either the mutation c.2299delG or c.2276G>T. Green arrowheads indicate fragments cut by the restriction enzyme MlsI.