Hossain Kazi A, Nierzwicki Lukasz, Orozco Modesto, Czub Jacek, Palermo Giulia
Department of Bioengineering , University of California Riverside, Riverside, United States.
Department of Physical Chemistry, Gdańsk University of Technology, Gdańsk, Poland.
Elife. 2025 Feb 10;13:RP102538. doi: 10.7554/eLife.102538.
xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.
xCas9是CRISPR-Cas9基因组编辑系统的一种经过改良的变体，经过改造以提高特异性并减少不必要的脱靶效应。xCas9如何通过识别一系列替代的原间隔相邻基序（PAM）序列而忽略其他序列来扩展Cas9的DNA靶向能力尚不清楚。在这里，我们阐明了xCas9扩展PAM识别的分子机制，并为扩展DNA靶向提供了关键见解。我们证明，虽然野生型Cas9通过其相互作用的精氨酸二元组的刚性来强制严格的鸟嘌呤选择，但xCas9在R1335中引入了灵活性，从而能够选择性识别特定的PAM序列。这种增加的灵活性赋予了明显的熵偏好，这也提高了对经典TGG PAM的识别。此外，xCas9在早期进化周期中增强了与替代PAM序列的DNA结合，而在最终进化周期中则倾向于与经典PAM结合。这种双重功能突出了xCas9如何拓宽PAM识别，并强调了微调PAM相互作用裂隙的灵活性作为扩展CRISPR-Cas系统DNA靶向潜力的关键策略的重要性。这些发现加深了我们对xCas9中DNA识别的理解，并可能适用于其他具有类似PAM识别要求的CRISPR-Cas系统。
