Sangamo Therapeutics, Inc., Richmond, CA, USA.
Nat Biotechnol. 2019 Aug;37(8):945-952. doi: 10.1038/s41587-019-0186-z. Epub 2019 Jul 29.
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.
基因编辑技术凭借其高效基因组编辑的能力而备受关注。然而，这些试剂的特异性仍然是一个问题，特别是在治疗应用中，因为脱靶切割可能会产生潜在的诱变后果。在这里，我们开发了一种提高锌指核酸酶 (ZFN) 特异性的方法，通过工程改造 FokI 催化结构域来减缓切割速度，这应该可以选择性地降低低亲和力脱靶位点的活性。对于三个 ZFN 对，我们在 FokI 结构域中设计了单个残基取代，这些取代保留了全靶标活性，但在脱靶插入缺失方面的活性降低了 3000 倍。通过将这种方法与降低锌指亲和力的取代相结合，我们开发了针对 TRAC 基因座的 ZFN，它们介导 T 细胞中的 98%基因敲除，而在检测背景约为 0.01%的情况下，没有检测到脱靶活性。我们预计，这种方法和我们报告的 FokI 变体将能够常规地生成具有可检测脱靶活性的基因编辑用核酸酶。
