Enhancing gene editing specificity by attenuating DNA cleavage kinetics


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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Fig. 1: Identification of FokI substitutions that improve ZFN on-target cleavage preference.
Fig. 2: Global suppression of off-target cleavage by the I479Q and Q481A FokI domain variants.
Fig. 3: Improving ZFN cleavage specificity via removal of a nonspecific DNA contact in the zinc finger repeat.
Fig. 4: Development of ZFNs for highly efficient modification of TRAC in T cells with no detectable off-target effects.

Data availability

Illumina sequencing data underlying all key experiments have been deposited in the NCBI Sequence Read Archive under accession code PRJNA540312.

Code availability

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.

Author information

J.C.M. and E.J.R. designed experiments, supervised experiments, analyzed data and wrote the paper. F.F., D.P.P. and P.L. designed experiments, performed experiments and analyzed data. A.R., D.E.P. and L.Z. designed experiments, supervised experiments and analyzed data. G.L. designed experiments. D.A.S. and Y.R.B. wrote custom computer code. S.J.H. supervised experiments. C.B.P., D.F.X., H.W.R., N.A.S., S.C.L., T.W. and Y.Z. performed experiments.

Correspondence to Edward J. Rebar.

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All authors are full-time employees of Sangamo Therapeutics.

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Supplementary Information

Supplementary Figs. 1–15, Supplementary Tables 1–20 and Supplementary Notes 1 and 2

Reporting Summary

Supplementary Table 21

Detailed information for high-sensitivity OT1 indel assay results for K562 cells treated with Q481A AAVS1 ZFNs or GFP

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