美国Sangamo Therapeutics公司的Edward J. Rebar团队，通过降低DNA剪切活性实现对基因编辑特异性的增强。相关论文于2019年8月发表在《自然—生物技术》杂志上。

研究人员开发出一种增加锌指核酸酶的特异性(ZFN)的方法，通过利用减慢剪切的臂加工Fokl催化结构域，从而选择性降低其在低亲和力脱靶位点的剪切活性。研究人员在三对ZFN中替换了FokI结构域的单个位点，从而对正确位点保持完整剪切活性，但对脱靶性插入缺失降低了近3000倍。通过结合这种方法与降低锌指亲和力的替换方法，研究人员开发了特异性靶向TRAC基因座的ZFN，其能够在T细胞中实现98%的敲除效率但在0.01%左右的背景下检测不到脱靶活性。研究人员认为这种方法以及他们报道的Fokl变体，能够常规产生用于基因编辑的核酸酶但检测不到脱靶活性。

据介绍，工程核酸酶因为其高效的基因组编辑能力获得了广泛认可。然而，考虑其脱靶剪切引起的潜在突变，这些工具的特异性仍然是一个问题，特别是对于治疗应用而言。

附：英文原文

Title: Enhancing gene editing specificity by attenuating DNA cleavage kinetics

Author: Jeffrey C. Miller, Deepak P. Patil, Danny F. Xia, Charles B. Paine, Friedrich Fauser, Hunter W. Richards, David A. Shivak, Yuri R. Bendaa, Sarah J. Hinkley, Nicholas A. Scarlott, Stephen C. Lam, Andreas Reik, Yuanyue Zhou, David E. Paschon, Patrick Li, Tenzin Wangzor, Gary Lee, Lei Zhang, Edward J. Rebar

Issue&Volume: Volume 37 Issue 8

Abstract: 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.

DOI: 10.1038/s41587-019-0186-z

Source: https://www.nature.com/articles/s41587-019-0186-z