Figure 2

In vivo nuclease activity of the three nuclease architectures in mammalian cells. (A) Schematic representation of nuclease-mediated gene inactivation via the error-prone NHEJ pathway. (B) Size distribution of the deletion events induced at the endogenous locus by the TtH architecture. Loci presenting at least 20 events were taken into account to generate the figure. Error bars denote s.d. Student t test performed to compare deletion patterns induced by TtT and TtH architectures showed no statistical difference (p-value = 0.3852). (C) Same as for (A) but for the HtH nuclease architecture. One locus presenting at least 20 events was used. (D) Same as for (A) but for the TtT nuclease architecture. (E) Representation of the localization of the deletion center for the three architectures. The Gaussian curves having the same mean and variance of deletion centers for each of the three TtH, HtH, and TtT architectures are represented. The areas under the curves have been normalized to 1. t-test, p-value = 0.00155 with respect to the TtT architecture. For the TtT and HtH architectures, due to the odd number of nucleotides present in their spacer (15 or 25 respectively), we arbitrary chose to place the center of the spacer at 8 or 13 bp, explaining the shift of the deletion center close to -1. Data from 3 loci (DMD, FUT8 and RAG) were used for the HtH and TtH architectures. Data from 10 loci (APC, MLH, CD52, NR3C3, LIG4, BBC3, NR3C2, M2K, PPARD, ERBB2) were used for the TtT architectures.