Supplementary Figure 4: Detection of SNPs with TALEs.

(a) We analyzed sequencing data from 16 clones of Mus spretus minor satellite using Tandem repeat finder (http://tandem.bu.edu/trf/trf.html)³¹ and found that the minor satellite sequence has a basic higher order repeat of 120 bp, composed of two 61 bp (top) and 59 bp (bottom) monomers. The sequence conservations between sequence reads of these monomers are shown as the height of a stack of letters at each base position. The sequence data is provided in Supplementary Table 2. (b) Alignment of minor satellite sequence of Mus musculus (GenBank: Z22170.1) and Mus spretus is shown. Asterisks mean the position of sequence differences. (c) Alignment of minor satellite sequence of Mus musculus (top) and Mus spretus (bottom). Asterisks indicate the positions of SNPs between them. Arrows (mi01-mi09) indicate targeting sequence of TALEs. mi01-06 are directed against Mus spretus sequence. mi07 was designed against Mus musculus sequence. mi08 and mi09 are directed against the identical sequences. (d and e) Alignments of minor satellite sequence of Mus musculus and Mus spretus are indicated at the top, together with the targeting sequence used to design the TALE-VP64 (arrows). 293T cells were transfected with the indicated TALE-VP64 plasmids and the firefly reporter plasmid containing the TALE targeting sequence. Relative luciferase activity representing the binding affinity of TALEs is shown at the bottom. The mean ± s.d. of three independent biological replicates is shown. (d) Binding affinity of TALEs targeting MinSat sequence of Mus musculus and Mus spretus harboring two SNPs is shown. TALE specifically binds to corresponding origin of MinSat, indicating these TALEs distinguish two nucleotide differences. (e) Binding affinities of TALEs against mi08 and mi09 harboring one SNP is shown. Both TALEs bound specifically to MinSat of Mus spretus, suggesting that TALEs can recognize a single nucleotide difference. (f) Representative images of the pericentromeric region of mitotic chromosomes derived from Mus musculus (top) and Mus spretus (bottom) in SF1 cells expressing TALE-mClover (green) recognizing the MajSat sequence and TALE-mRuby2 (magenta) recognizing the indicated MinSat sequence. Large fluorescent foci of TALE-mClover were used as reference to distinguish Mus musculus or Mus spretus chromosomes, respectively. Target genome and number of SNPs for indicated TALEs are indicated above and below the images, respectively. (g) Representative images of mitotic chromosome of SF1 cells expressing the indicated TALE-mRuby2 against MinSat sequence (red) together with the TALE-mClover against the MajSat sequence (green). Scale bar, 2 μm.