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Abstract
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.
Highlights
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences
If the break is repaired by mutagenic non-homologous end joining (NHEJ), the insertions or deletions that often result can lead to frame shifts and functional knockouts[2]
If the break is repaired by homology-directed repair (HDR), the sequence can be rewritten if an appropriate donor template of DNA is provided[3,4]
Summary
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. Algorithms that allow the user to choose a specific target sequence based on its reduced risk of off-target activity have been developed[31,32,33] These approaches limit therapeutic applications that require precise modification of sequences with high degrees of homology to other regions of the genome. Prior ZFN engineering for in vivo application by both bacterial one[38] and two-hybrid[39,40] assays have been primarily limited to pools of 50-GNN-30 binding zinc fingers and do not present a counterselective element that would produce zinc fingers able to discriminate between closely related sequences This new complete zinc-finger pool set coupled with a system that provides designer nucleases, zinc finger or otherwise, that can distinguish between two closely related sequences would be of great utility
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