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A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases

Shimin Lim1, Yin Wang23, Xueyao Yu4, Yian Huang5, Mark S Featherstone1 and Karuna Sampath123*

Author Affiliations

1 School of Biological Sciences, 50 Nanyang Avenue, Nanyang Technological University, Singapore 639798

2 Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604

3 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543

4 School of Applied Science, Temasek Polytechnic, 21 Tampines Avenue 1, Singapore 529757

5 NUS High School of Mathematics and Science, 20 Clementi Avenue 1, Singapore 129957

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Genome Biology 2013, 14:R69  doi:10.1186/gb-2013-14-7-r69

Published: 1 July 2013

Additional files

Additional file 1:

Figures S1 to S5 and Tables S1 to S3. Figure S1: (A) graph showing percentage cutting efficiency of single TALEN pairs targeting egfp, sqt and cyc, in comparison to double TALEN pairs, as determined by sequencing (percentage 5'-3' complete deletions amongst all sequenced alleles for double TAL pair injections are shown as purple bars). (B) Graph showing frequency of different mutant alleles in double TALEN pair-injected embryos for egfp, sqt and cyc. Deletions in the 5' site alone, 3' site alone, complete 5'-3' deletions, and incomplete 5'-3'deletions (which are larger than individual 5' alone or 3' alone deletions but smaller than complete 5'-3' deletions) were observed. (C) Table showing frequency of mutations induced by double nuclease pair injections as a percentage of total number of alleles. (D) Table showing frequency of mutations induced by single nuclease pair injections. (E-J) Alignment of egfp, sqt and cyc sequences from single or double nuclease pair injected embryos showing 5' only, 3' only, and incomplete or complete 5'-3' deletions. For double-nuclease pair injections, the target site of the 5' pairs is highlighted in yellow and the 3' pairs in green. Insertions are highlighted in blue and deletions are indicated with red dashed lines. Numbers in the middle of the alignment indicate the number of intervening gaps and bases. Single nucleotide substitutions are highlighted in magenta. Nature and extent of mutations, and frequency of alleles observed >1 (× n) are shown to the right of the alignments. Figure S2: representative phenotypes observed at 24 h in embryos injected with sqt or cyc TALENs. At high doses, the proportion of abnormal embryos increases. Figure S3: (A) Alignment of cyc sequences showing TSS deletions in embryos from F0 founders, compared to wild-type cyc. Insertions are indicated in red, and gaps are shown by dashed lines. Letter suffixes (for example, 1A and 1B) represent different alleles from the same founder. Eight founders injected with cyc TALENs transmitted complete deletion of the intervening sequences, some of which also show insertion events. Founder F0-4, shows a larger deletion that extends beyond the 3' end of the cyc3TAL target site. Genomic coordinates on chromosome 12 for wild-type cyc are indicated for the regions shown. (B) Alignment of cyc sequence of F0-10 to wild-type cyc shows a deletion (dashed lines), accompanied by an inversion (yellow highlight) and insertion (red font). Figure S4: (A) Alignment of sqt sequences showing the whole-locus and TSS deletions in embryos from F0 founders, compared to wild-type sqt. Insertions are highlighted in red font, and gaps are indicated with dashed lines. All six founders for sqt5TAL/sqt3TAL showed embryos with the 2.1 kb whole locus deletion. (B) For sqt5TAL/sqtZFN2, founder 1 (F0-1) showed embryos with the intervening sequences excised, whereas founder 2 (F0-2) sequences indicate that only sqt5TAL was active. Genomic coordinates on chromosome 21 for wild-type sqt are indicated for the regions shown. TALEN and ZFN target sites are indicated. Figure S5: alignment of sqt sequences showing the sqtsg27 TSS deletion (A), sqt sg32 whole-locus deletion (B), and sqtsg7 ZFN (C) mutations in comparison to wild-type sqt. Insertions are highlighted in red font, and gaps are indicated with dashed lines. TALEN and ZFN target sites are indicated. Table S1: list of target sites for TALENs and ZFNs. Table S2: list of primers for genotyping, sequencing, and T7E1 assays. Table S3: list of primers for detecting expression of sqt, rnf180, htr1ab, eif4ebp1 and act.

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