Kleinstiver Benjamin P, Wang Li, Wolfs Jason M, Kolaczyk Tomasz, McDowell Brendon, Wang Xu, Schild-Poulter Caroline, Bogdanove Adam J, Edgell David R
Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1.
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science, Ithaca, New York 14853.
G3 (Bethesda). 2014 Apr 16;4(6):1155-65. doi: 10.1534/g3.114.011445.
Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5'-CNNNG-3'. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints.
工程化核酸酶能够以位点特异性方式进行切割，从而实现对复杂基因组的精确编辑。在此，我们将归巢内切酶I-TevI的小的、序列耐受性单体核酸酶结构域与转录样激活效应因子（TALEs）融合，构建了单体Tev-TALE核酸酶（Tev-mTALENs）。利用PthXo1 TALE支架优化Tev-mTALEN结构，我们发现在基于酵母的实验中，TALE上N端融合点的选择对活性有很大影响，并且所使用的接头长度影响了由基序5'-CNNNG-3'指定的TALE结合位点与I-TevI切割位点的最佳间距。通过检测该基序所有64种可能序列变体上的活性，我们发现，在Tev-mTALEN环境中，I-TevI在切割位点处更倾向于富含A/T的三联体而非富含G/C的三联体。对将CNNNG基序与TALE结合位点分隔开的DNA间隔区中的核苷酸需求进行分析，结果显示对序列变异具有显著但不完全的耐受性。Tev-mTALENs在HEK 293T细胞的游离型靶标上表现出强大的诱变活性，这与特异性切割后进行非同源末端连接修复一致。我们的数据证实了Tev-mTALENs作为基因组编辑工具的适用性，但也突出了DNA间隔区和切割位点的核苷酸偏好，这些偏好虽然增强了特异性，但确实带来了一定的靶向限制。
