Department of Biomedical Engineering, Tufts University, Medford, MA 02155.
Proc Natl Acad Sci U S A. 2024 Jan 9;121(2):e2308415120. doi: 10.1073/pnas.2308415120. Epub 2023 Dec 27.
Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.
噬藻体 S-2L 病毒的基因组 DNA 由 2-氨基腺嘌呤（Z）、胸腺嘧啶（T）、鸟嘌呤（G）和胞嘧啶（C）组成，形成遗传字母 ZTGC，违反了沃森-克里克碱基配对规则。Z 碱基在 2 号位上有一个额外的氨基，允许在 DNA 链中与胸腺嘧啶形成第三个氢键。在这里，我们探索并扩展了这种非沃森-克里克碱基配对在蛋白质表达和基因编辑中的应用。体外产生的 ZTGC-DNA（Z-DNA）和 ZUGC-RNA（Z-RNA）都表现出可检测的兼容性，并可以在哺乳动物细胞中解码，包括 细胞。Z-crRNA 可以通过非沃森-克里克碱基配对引导 CRISPR 效应物 SpCas9 和 LbCas12a 切割特定的 DNA，并与 A-crRNA 相比提高切割活性。Z-crRNA 还可以允许在人类细胞中进行有效的基因和碱基编辑。总之，我们的结果为优化基因编辑治疗的 DNA 或 RNA 有效载荷的潜在策略提供了帮助，并为理解天然 Z-DNA 基因组提供了思路。
