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Shown by Bat1-FokI was 35 cleavage (11 bp spacer) while dTALEBat1mimic -FokI had a maximum efficacy of 86 cleavage (19 bp spacer; Figure 4). That dTALEBat1mimic -FokI showed greater flexibility with respect to spacer length may relate to the previously optimized architecture employed (18). TALEN architecture is known to play a decisive role in spacer preference (23). Similarly, alternative Bat1
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Ckground (Figure 5B). This does not match expectations based on TALEs where only the cryptic N- but not the cryptic C-terminal repeats are essential for DNA binding (26). By contrast, our results suggest that the cryptic C-terminal Bat1 repeat +1, in contrast to the corresponding cryptic TALE repeat +1, makes an unexpectedly strong contribution to activity and thus should be retained for the creat
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E S6) in order to determine the spacing between the two target sites that would result in the highest activity of the Bat-FokI fusion proteins. As a negative control, we tested a template with a control sequence instead of the Bat1 target sites. Bat1-FokI and dTALEBat1mimic -FokI were expressed in vitro and equal volumes of reaction product were incubated with the target DNA. After 3 h at 37 C the
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Rminal repeats are typically inflexible, though alternative repeat -1 modules have recently been described (24,25). We tested acBat1 deletion derivatives to test if this paradigm applies to Bat1. First, we tested variants of acBat1 lacking 2 ( 18?0), 4 ( 16?0), 6 ( 14?0) or 8 ( 12?0) core repeats (Figure 5A and Supplementary Figure S10). The later half of repeat 20 and repeat +1 were retained in e
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Rminal repeats are typically inflexible, though alternative repeat -1 modules have recently been described (24,25). We tested acBat1 deletion derivatives to test if this paradigm applies to Bat1. First, we tested variants of acBat1 lacking 2 ( 18?0), 4 ( 16?0), 6 ( 14?0) or 8 ( 12?0) core repeats (Figure 5A and Supplementary Figure S10). The later half of repeat 20 and repeat +1 were retained in e
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Lymorphism in the native Bat1 as well as being relevant for the creation of Bat1 derivatives with novel specificity (dBats). We hypothesize that nonRVD polymorphisms may have two functionally relevant, non-mutually-exclusive, effects. (i) The formation of unique but functionally equivalent repeat interfaces that stabilize the superhelical structure formed by tandem-arranged repeats (4,5) (superstr
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Ats are near identical and repeat order does not change the interface between repeats. Given the numerous nonRVD polymorphisms between Bat1 repeats, deletion or insertion of core repeats will always create novel repeat interfaces and should be experimentally validated before use in downstream applications. We next tested acBat1 derivatives where the 82 residues N-terminal of core repeat 1 (acBat1
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Pplementary Figure S11. In the repeat switch whole repeats, including their native RVDs, were exchanged. This creates new interfaces between repeats but leaves RVDs in their native repeat context. If the superstructural hypothesis is correct then the repeat switch is likely to modify evolved repeat interfaces possibly yielding less active DNA-binding proteins. In the RVD switch it is only the RVDs