CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.

CRISPR–Cas9 是一种多功能 RNA 引导的基因组编辑工具。在这里，我们证明了 CRISPR RNA (crRNA) 中的 RNA 核苷酸部分替换为 DNA 核苷酸，可以在人类细胞中进行有效的基因编辑。当与 crRNA 和 sgRNA 以及多个引导序列一起使用时，这种部分 DNA 替换策略保留了目标活性。部分 DNA 替换也适用于另一种 CRISPR 系统 Cpf1 的 crRNA。我们发现，通过对脱靶切割的集中分析、错配耐受性的测量和脱靶位点的全基因组分析，引导序列中的部分 DNA 替换显着减少了脱靶基因组编辑。以Cas9-sgRNA复合物的结构为指导，crRNA的3′端大部分可以被DNA核苷酸取代，5-和3′-DNA取代的crRNA可以实现高效的基因组编辑。由 DNA-RNA 嵌合体引导的 Cas9 可能提供一种通用策略，以降低人类细胞中的成本和脱靶基因组编辑。