Easy And Rapid Preparation Of Stable CRISPR-Cas9-sgRNA Complex And Its Application

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated-protein 9(Cas9)genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids.The widely used Streptococcus pyogenes Cas9(SpCas9)can be targeted to a 20 bp specific DNA sequence by an associated complementary guide RNA(gRNA),provided that a protospacer adjacent motif(PAM)of the form NGG is present.The CRISPR-Cas9 based genetic technologies,in particular as genome-editing tools,have been extensively applied to biomedical area with great promise to revolutionize the treatment of genetic diseases..In spite of in vivo applications,the CRISPR-Cas9 has also been employed as in vitro molecular cloning tools.For instance,a technique named CATCH utilized Cas9 nuclease to excise the target genome segment up to 100 kb and then ligate it to the cloning vector in a single step.Another creative technique called DASH can harness Cas9 to effectively deplete unwanted sequence from DNA libraries prior to sequencing.In these techniques,Cas9 functioned as a ribonucleprotein complex(RNP),in which the specific negative single guide RNA molecules(sgRNAs)were bound to the positively charged Cas9.The strategy currently available to prepare Cas9 RNP needs to obtain Cas9 enzyme and sgRNAs separately and then assemble them in vitro.Cas9 is typically purified from E.coli by recombinant expression,whereas sgRNAs are constructed by in vitro transcription with T7 RNA polymerase.By transcription method,it requires multiple steps to gain up-to-standard sgRNAs.So it is not appropriate for simple and rapid preparation of Cas9 RNP.which limits their board applications.Herein,we creatively developed an efficient method for direct preparation of Cas9 RNP from E.coli.Specifically,we harnessed an engineered cold-shock vector to realize co-expression and self-assembly of Cas9 and sgRNAs in E.coli cells.After one-step affinity purification,we obtained ready formed RNP with purity over 80%.Unexpectedly,such kind of Cas9 RNP is super-stable that remained full endonuclease activity at 4? for three months.Furthermore,the stable RNP can be conveniently stored at-20? for up to half a year till use.Strikingly,our new method greatly simplified the course of RNP constructions.In a standard process,one specific RNP can be readily prepared within three days from constructed plasmid to the target RNP,which significantly reduces the cost of production.Collectively,our method enables simple and rapid production of Cas9 RNP.We next explored the potential applications of super-stable Cas9 RNP above,with particular focus on molecular cloning tools in vitro.Initially,we constructed a series of RNPs instead of restriction endonucleases to efficiently cleave vectors at multiple cloning site(MCS).Next,we simultaneously assembled multiple gene fragments with almost 100%fidelity by RNP cleavage combined with a T5 exonuclease-assisted cloning method.Specifically,for a three-fragment assembly,only a single RNP was required for cutting DNA fragments off from three donor plasmids,as well as obtaining a linear vector from one acceptor plasmid,respectively.Lastly,we designed a cyclic assembly method termed Cas-Brick for multi-round assembly of several gene fragments using combinatorial Cas9 RNPs.For instance,with two rounds of action by four pairwise RNPs,nine individual DNA fragments with an overall length of 9 kb were seamlessly assembled.This RNP-based method can fully bypass PCR steps where mutations might be introduced,thereby achieving scarless assembly of multiple gene fragments with high fidelity.