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. Author manuscript; available in PMC: 2014 Oct 1.

Published in final edited form as: Nat Methods. 2014 Feb 16;11(4):429–435. doi: 10.1038/nmeth.2845

(A) Architecture of a TALEN. A TALEN monomer contains an N-terminal domain (blue) followed by an array of TALE repeats (brown), a C-terminal domain (green), and a FokI nuclease cleavage domain (purple). The 12^th and 13^th amino acids (the RVD, red) of each TALE repeat recognize a specific DNA base pair. Two different TALENs bind their corresponding half-sites, allowing FokI dimerization and DNA cleavage. The C-terminal domain variants used in this study are shown in green. (B) A single-stranded library of DNA oligonucleotides containing partially randomized left half-site (L), spacer (S), right half-site (R) and constant region (thick black line) was circularized, then concatemerized by rolling circle amplification. The concatemerized double stranded DNA (double arrows) contained repeated target sites with L′ S′ R′ representing the reverse sequence complement of L S R. (C) The concatemerized DNA libraries of mutant target sites were incubated with an in vitro-translated TALEN of interest. Cleaved library members were blunted and ligated to adapter #1. The ligation products were amplified by PCR using one primer consisting of adapter #1 and the other primer consisting of adapter #2–constant sequence, which anneals to the constant regions. From the resulting ladder of amplicons containing a half-site with an integral number (n) of repeats of a target site (represented by brackets), amplicons corresponding to 1.5 target-sites in length were isolated by gel purification and subjected to high-throughput DNA sequencing and computational analysis (Supplementary Algorithms).

(A) Architecture of a TALEN. A TALEN monomer contains an N-terminal domain (blue) followed by an array of TALE repeats (brown), a C-terminal domain (green), and a FokI nuclease cleavage domain (purple). The 12^th and 13^th amino acids (the RVD, red) of each TALE repeat recognize a specific DNA base pair. Two different TALENs bind their corresponding half-sites, allowing FokI dimerization and DNA cleavage. The C-terminal domain variants used in this study are shown in green. (B) A single-stranded library of DNA oligonucleotides containing partially randomized left half-site (L), spacer (S), right half-site (R) and constant region (thick black line) was circularized, then concatemerized by rolling circle amplification. The concatemerized double stranded DNA (double arrows) contained repeated target sites with L′ S′ R′ representing the reverse sequence complement of L S R. (C) The concatemerized DNA libraries of mutant target sites were incubated with an in vitro-translated TALEN of interest. Cleaved library members were blunted and ligated to adapter #1. The ligation products were amplified by PCR using one primer consisting of adapter #1 and the other primer consisting of adapter #2–constant sequence, which anneals to the constant regions. From the resulting ladder of amplicons containing a half-site with an integral number (n) of repeats of a target site (represented by brackets), amplicons corresponding to 1.5 target-sites in length were isolated by gel purification and subjected to high-throughput DNA sequencing and computational analysis (Supplementary Algorithms).