PT - JOURNAL ARTICLE AU - Li, Wenqiang AU - Li, Shuntang AU - Qiao, Jie AU - Wang, Fei AU - Liu, Yang AU - He, Ruyi AU - Liu, Yi AU - Ma, Lixin TI - Direct preparation of Cas9 ribonucleoprotein from <em>E. coli</em> for PCR-free seamless DNA assembly AID - 10.1101/328468 DP - 2018 Jan 01 TA - bioRxiv PG - 328468 4099 - http://biorxiv.org/content/early/2018/06/07/328468.short 4100 - http://biorxiv.org/content/early/2018/06/07/328468.full AB - CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.