CRISPR Mediated Large DNA Fragment Cloning

Microbial Natural products(NPs)are major sources of drugs and chemicals,and most NPs are produced by biosynthetic gene clusters(BGCs).In microorganisms,genes responsible for the synthesis and regulation of NPs are usually compressed into large DNA fragments of tens to hundreds of kb.To mine NPs with novel structures,the first and crucial step is to clone large BGCs with high-accurately and efficiency.Conventional DNA cloning is based on PCR-based fractional amplification of the gene of interest and sequential assembly using DNA assembly methods.However,the existing DNA clone methods have some problems,such as time-consuming,low efficiency,complex operation,and so on.Therefore,the development of novel large DNA fragment cloning methods with high-efficiency,accurate,and easy-to-operation remains a necessity.The CRISPR/Cas9 system only requires Cas9 nuclease and engineered sgRNA to target DNA with high specificity.Due to its high specificity and programmability,it has been widely used in various fields such as gene editing,cell imaging,and medical treatment.In this study,we developed a large DNA fragment cloning technology based on the CRISPR/Cas9 system to avoid circumventing the restriction site restriction of traditional methods.Firstly,high-efficiency editing targets are screened by gRNA design tools,which alleviates low targeted cleavage efficiency at some sites,reduces off-target effects,and saves screening costs.Secondly,the CRISPR/Cas9 system was used to cut genomic DNA directly,and the target DNA fragments were directly obtained from the genome avoiding PCR amplification limitations and mutations.We used enzymatic and phenol chloroform to prepare high-quality and high-molecular-weight DNA for large DNA fragment cloning.In this study,we used Blue-White Screening for positive transformants screen and positive rate calculation.Two gRNAs are designed to target genome DNA,and DNA fragments are assembled by NEBuilder.We successfully established CRISPR/Cas9 mediated large DNA fragment clone method.To achieve an efficient DNA clone,we selected a 15 kb DNA fragment containing the LacZ gene as the target to optimize the restriction enzyme digestion system,genome content,homology arm length,and other conditions.The results showed that when the molar ratio of Cas9:gRNA:genome was between 5000:5000:1～10000:10000:1,and the molar ratio of insert:vector was 1:1,the cloning efficiency was close to 100%.Then,we used this method to clone30-100 kb DNA fragments.When the size of DNA fragment below 50 kb,and the cloning efficiency is nearly 100%.We successfully clone 77 kb DNA fragment with 46% efficiency.And then,the method was also successfully applied to S.coelicolor,and a 40 kb DNA fragment was successfully cloned with a positive rate of 93%.In this study,we established a system for large DNA fragment clone based on the CRISPR/Cas9 system,a high-quality genomic DNA,and the NEBuilder seamless cloning method.Compared with other large fragment DNA cloning techniques,this method has the characteristics of simplicity,high operability,high efficiency,and time-consuming.