Figure 1.
The design parameters of active 15-RVD GoldyTALEN pairs used in this study. Each TALEN arm consists of a DNA binding domain with 15-RVDs (14.5 TALE repeats), corresponding to 15-nucleotides DNA binding sequence proceeded by a 5′ T nucleotides and a 13 to 20 bp spacer in between 2 arms containing a restriction recognition sequence to assay activity. *Initial design parameter was spacer length between 11–20 bp and only the two inactive pairs, NPM1B P1 and P2 have spacers shorter than 13 bp.
Figure 2.
Synthesis of 15-RVD GoldyTALENs.
A schematic diagram showing the assembly of 15-RVD GoldyTALENs by the Golden Gate method described earlier [17]. The RCIscript-GoldyTALEN backbone, all other component plasmids (as Golden Gate TALEN kit 2.0), and the 256 pFUS_B4 clones are distributed through Addgene. *XX denoted either NI, HD, NN or NG.
Figure 3.
In vivo activity of 15-RVD GoldyTALENs.
(A) In vivo activity (% of chromosome conversion at somatic level) of 15-RVD TALEN pairs. Results shown were averages of 3 separate experiments analyzing groups of 10 embryos. Error bars represent the standard error of the means. (B) Representative results of RFLP screening assay after injection of IDH1 P1 and GFP(GM2) P1. Open arrowheads indicate bands from completely digested WT PCR product and closed arrowheads represent uncut PCR product with small indels. *Marks single embryos with bi-allelic conversion.
Figure 4.
Importance of the spacer length and the 5′ T nucleotide in GoldyTALEN activity.
(A) Modifications of NPMB1 P1 to LS and RS for longer spacers. (B) Both modified NPMBP1 TALEN pairs (NPM1B P1 LS and RS) showed significant increase in in vivo activity compared with the original shorter spacer design. (C) Modifications of IDH1 P1 and JAK2A P1 to remove the 5′ upstream T nucleotide at one of the TALEN arms. (D) Both modified TALEN pairs (IDH1 P1 RM and JAK2A P1 LM) showed significant reduction in in vivo activity compared with the original designs. Open arrowheads indicate bands from completely digested WT PCR product and closed arrowheads represent uncut PCR product with small indels. WT: wild-type; T: TALEN pair injected. Representative and average results of RFLP screening in 3 separate experiments analyzing group of 10 embryos are shown in the gel photo and graph, respectively. Error bars represent the standard error of the means.
Table 1.
Correlation of spacer lengths and somatic activities of 15-RVD GoldyTALENs.
Figure 5.
Deletion of large genomic fragments with two pairs of TALEN.
(A) Schematic diagram showing the strategy of introducing large genomic deletions at flt3 and jak2a loci using two TALEN pairs. Green and purple arrows represent primer pairs used for PCR screening of corresponding large deletion. WT: wild-type; T: TALEN pairs injected. (B) Sequences of germline-transmitted large deletions in F1 embryos. Sequence of a single mutant F1 embryo from each founder is shown. Underlined are TALEN binding sites. Sequences in blue represent insertions.
Figure 6.
Somatic efficacy of 15-RVD TALEN compared with other reported TALEN scaffolds, ZFN and CRISPR/Cas9.
Plot of somatic mutation rates in zebrafish genome editing using ZFNs [8], [32], TALENs [25], [29], [30], [32] and CRISPR/Cas9 [46] from representative publications together with 15-RVD TALENs from this study. *Only Homo-dimeric TALENs were compared. †Only 15-RVD TALENs with spacer lengths between 13–20 bp and a T nucleotide preceding the TALEN binding site were included.