The discovery^1,2 and the pioneering applications³ of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)^4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure^5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches⁷. This process is still poorly understood on a mechanistic level^8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction.

中文翻译：

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CRISPR-Cas Cascade 复合体形成 R-loop 的能量景观

CRISPR-Cas 效应复合物的发现^1,2和开创性应用³提供了强大的基因编辑工具。效应复合物通过其 CRISPR RNA (crRNA) ^4,5的互补性被引导至目标基因组位点。双链 DNA 目标的识别通过 DNA 展开和 crRNA 与 DNA 目标链之间的碱基配对进行，从而形成 R 环结构^5,6。完整的 R 环形成是随后 DNA 切割的先决条件。虽然 CRISPR-Cas 技术易于使用、高效且用途广泛，但由于识别具有多重错配的非预期序列，脱靶效应阻碍了治疗应用⁷. 这个过程在机械层面上仍然知之甚少^8,9。特别是，缺乏对 R 环形成的能量学和动力学的深入了解，阻碍了对 R 环形成的直接建模以进行脱靶预测。