Enzymatical Tri-libraries Strategy: A Method To Generate CRISPR/Cas9 Libraries For Genome Wide Screening

Gene editing is one of the important technologies in bioengineering,and it is widely used in basic research,clinical treatment,genetic breeding,and ecological diversity.Recently,the novel artificial endonuclease CRISPR/Cas9 system has been used in ge ne editing,and it greatly improved the efficiency of gene editing.The CRISPR/Cas9 system has a high efficiency and accurate editing in the gene knock-out.At the same time,the CRISPR/Cas9 system has the advantages of easy operation and low cost in the application.It makes the CRISPR/Cas9 system as the basic technology of gene knockout library,especially in the whole genome wide high-throughput screening.At present,genome wide knockout library of mouse,human and other species has been constructed,which relying on artificial design and synthesis guide RNAs.Although the cost of CRISPR/Cas9 is lower than that of ZFN or TALEN,the large-scale high-throughput synthesis process,manual operation and cost are not negligible.On the other hand,the guide RNA design is severely constrained by the annotation of the genome of the species.The less species genome annotated,the less guide RNA coverage affected.It is critical to design one strategy to construct a highly efficient,non-synthetic,high-coverage guide RNA library which CRISPR/Cas9 system mediated.Recently,there are some reports on the strategy of enzyme construction guide RNA libraries.However,there are still some problems to be solved in these research,including the vector of knock-out library,the source of guide RNA fragments,and the limitation of screening application.In response to these problems and the limitations,a new construction strategy of CRISPR/Cas9-mediated genome wide knockout library had been designed and proposed —— "Enzymatical Tri-libraries strategy".According to the characteristics of Type II and Type IIS restriction enzymes,guide RNA fragments had been producted by digesting genome DNA with these restriction endonuclease and the guide RNA library had been constructed.T his strategy not only avoids the influence of bioinformatics annotation in the design process of guide RNA,but also avoids the guide RNA synthesizing.The cost of guide RNA construction had been greatly decreased,because the source of guide RNA fragments are genome DNA.This strategy can be applied to any species-only need to extract the genome DNA of the species.such a general-purpose construction strategy,it provide a database construction strategy for each species of research,and rely on the knock-out library,the biological process of each species Mechanism-related gene mining and revealing the molecular mechanisms behind biological processes.The "Enzymatical Tri-libraries Strategy" is as follows:(1)Construction of the first-level library f.Msp I library.The genome was digested by Msp I.The recognition site CCGG contained the guide RNA-related PAM sequence CGG.The digested products were ligated into the dephosphorylated f.Msp I vector.The successful vector was characterized by: After Msp I digestion product DNA fragments being ligated into the vector,the Mme I and Mly I recognition sites are present on the vector adjacent to both ends of these fragments.A primary library named "f.Msp I library" had been constructed.(2)biotinylated PCR amplification.The f.Msp I library was amplified by biotin-labeled primers to obtain key regions in the f.Msp I library and labeled with biotin.(3)Mme I endonuclease treatment and specific fragment recovery.The biotinylated PCR product was digested with Mme I,and the DNA was cleaved at 20 bp downstream of the recognition site to produce a 20 bp DNA fragment derived from the genome.The digested product was separated by 3% agarose gel electrophoresis and recovered with 150 bp and 75 bp Fragments.(4)magnetic beads adsorption and Mly I endonuclease treatment.The recovered specific fragment was adsorbed by the magnetic beads and it digested by Mly I,and the 20 bp DNA fragment was cleaved from the excess DNA derived from f.Msp I vector,and the supernatant was subjec ted to Mly I inactivation treatment;(5)Secondary library PAM-F library construction.The inactivated Mly I product,a 20 bp genomic-derived DNA fragment,was ligated into the PAM-Fv4.0 vector to obtain the corresponding secondary library PAM-F library;(6)Tertiary library lenti library construction.The U6-g RNA region in the PAM-F library was ligated into the lenti CRSIPRv2 vector using Eco RI and Kpn I to obtain the corresponding Lenti library of the tertiary library.First,to verify the feasibility of the "Enzymatical Tri-libraries Strategy",we construced the knockout library for plasmid p EGFP-C1.The plasmid p EGFP-C1 was digested by Msp I and the primary library f.Msp I library had been constructed.The key region was amplified by biotinylation PCR.and then the PCR products were digested with Mme I to cleave the 20 bp DNA fragments which targeting p EGFP-C1 plasmid.The DNA fragments carrying the 20 bp fragment was adsorbed by the magnetic beads coated with streptavidin.The fixed end of DNA fragments were the vector-derived sequences,and the free end of DNA fragments were 20 bp genomic-derived sequences.It was digested with Mly I,and 20 bp DNA fragments were released into the supernatant for secondary library construction.The 20 bp DNA fragments were liga ted into the PAM-Fv4.0 vectors to obtain the corresponding PAM-F library,and the U6-g RNA region was ligated into the lenti CRISPR SP2 vector to construct a lenti library for subsequent screening.The knock-out libraries for plasmid p EGFP-C1 were packaged into lenti-viral and it would be used in infection the PKp G cell line,which expression EGFP protein,for screening.After virus infection and screening,EGFP-negative cells appeared in PKp G cell line,and the EGFP-negative rates were detected by FACS.The ratio of EGFP-negative cells was found to be about 50%(50.8%±14.8%,47.4%±1.2).%,48.2% ± 3.7%),significantly higher than the control group and the blank group.It demonstrated that the knockout library for p EGFP-C1 can be used for subsequent experimental screening.And it also further demonstrated the effectiveness of the strategy of constructing guide RNA library though the "Enzymatical Tri-libraries Strategy".In the process of constructing a knockout library against p EGFP-C1,the optimization steps were optimized for the actual implementation and corresponding results in the strategy.Including the comparison between the "add-linker strategy" and the "Tri-libraries Strategy",the validation of key restriction endonucleases and the adjustment of inadequate response,the optimization of f.Msp I library vector,the optimization of PAM-F library related vectors,etc.By optimizing the database building steps,the final efficiency of construction had been improved.Subsequently,according to the "Enzymatical Tri-libraries Strategy",the genome wide knockout library was constructed for mouse and pig genomes to verify the efficiency of this strategy applied to the whole genome knockout library.The genome DNA was digested by Msp I and a corresponding primary library f.Msp I library had been constructed.The key regions were amplified by biotinylation PCR,and the PCR products simultaneously subjected to Mme I endonuclease digestion.The fragmented DNA had been adsorbed by Magnetic beads.It was digested with Mly I to release a 20 bp DNA fragments.20 bp DNA fragments were ligated into the PAM-Fv4.0 vector to obtain a PAM-F library for the mouse,pig genome.And the obtained f.Msp I library and the PAM-F library were subjected to high-throughput sequencing.Sequencing results showed the target sites of mouse and pig genome knock-out libraries were widely distributed in whole genome,including coding regions,non-coding regions,regulary regions and intergenetic regions.It demonstrated that genome-wide knockout library for mammals can be successfully constructed according to the ?Enzymatical Tri-libraries Strategy?.The above results indicated that genome wide knockout libraries of different species can be constructed according to the "Enzymatical Tri-libraries Strategy".Compared with the previous library construction strategy,the advantage of this strategy is:(1)the "Enzymatical Tri-libraries Strategy" can avoid the limitation of guide RNA design caused by the incomplete biological information of the species;(2)the cost and operation amount can be greatly reduced during the database construction process;(3)The knockout libraries constructed by the strategy have higher coverage in the genome range,which leads to various sources of screening results,outside the coding region,and also targeting sites in the non-coding region and the cis-regulatory region;(4)these guide RNA libraries can also be applied to the epigenetic modification screening.However,the library construction strategy still has insufficient p erformance for subsequent optimization:(1)due to the limitation of the recognition site of Msp I,there were some omissions of other PAM sequences in the genome.(2)Due to the limitations of the CRISPR/Cas9 system,the knockout efficiency for non-coding regions is lower than the coding region knockout efficiency.(3)The genomes after digestion is highly complex,so the relevant operations need to be optimized during the construction of the f.Msp I library.In the future,in order to overcome these problem s,the construction strategy is further optimized to propose a genome-wide knockout library construction strategy with higher efficiency,more convenient operation,higher coverage and wider application range.