Figure 1. Strategy for the iterative in vivo selection of the entire SMN1 exon 7. (a) Selection procedure (diagram not to the scale). The pCI vector backbone is shown as a half‐circle. Exon 6 (E6), intron 6 (I6), partially random exon 7 (E7R), intron 7 (I7), and exon 8 (E8) sequences are marked; (b) Details of the second PCR amplification step and ligation. Primers B1 and B2 anneal to constant sequences in E6 and E8, respectively. B1 and B2 contain mutations marked in capitalized and underlined letters that create the BsaXI site and restore intronic sequences, respectively. Upon digestion with BsaXI, the intact E7R is released with the 3′ overhangs that correspond to the complementary intronic sequences of the 5′ overhangs of BsaXI‐digested pBxT2. The insertion of E7R into pBxT2 plasmid restores the minigene splicing cassette. For details of the primer sequences, refer to Table 1.

Figure 2. Comparison of splicing efficiency of different pools and selected sequences. (a) Comparative splicing patterns of different selected pools. The 333 and 279 bp products correspond to fully spliced and exon 7‐skipped products, respectively. The percentage of exon 7 skipping was calculated from the total value of exon 7‐included and excluded products. The abbreviations E6, E7, and E8 represent exon 6, exon 7, and exon 8, respectively. For details of the primer sequences, refer to Table 1; (b) Mutability of residues based on the results of in vivo selection. The values of −1 and +17.6 represent the absolutely conserved and the least conserved residues, respectively. The dotted horizontal lines show the cut‐off points with mutability values of +0.2 and −0.2, corresponding to the mutable and the conserved residues, respectively. Based on the stretches of the mutable and the conserved residues, the extended inhibitory context (Exinct), the 3′‐Cluster and the long conserved tract have been highlighted. Inhibitory nature of residues covering Exinct has been described in Ref. [ 4]. Consistent with the inhibitory nature of the 3′‐Cluster, deletions and mutations in this region promoted exon 7 inclusion in SMN2 [ 5]. Multiple mutations in the region of the conserved tract have been shown to cause exon 7 skipping in SMN1 [ 6, 9]. The exceptionally high mutability of position 54 is consistent with the dominant effect of A54G substitution on exon 7 inclusion [ 6]. Adapted from Ref. [ 5].