Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.
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Illumina sequencing data underlying all key experiments have been deposited in the NCBI Sequence Read Archive under accession code PRJNA540312.
Custom computer scripts used to perform the standard indel analysis and increased-sensitivity indel analysis can be found in Supplementary Note 2. Custom computer scripts used to automate more standard portions of the data analysis pipeline are available upon request.
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We thank M. Lal for performing preliminary experiments.
All authors are full-time employees of Sangamo Therapeutics.
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