Construction And Application Of Genome-wide CRISPR/Cas9 Library Mediated By Piggybac Transposon

The CRISPR library is an efficient mutagenesis tool for genome-wide screening and has been widely used in many research fields such as antiviral research,cancer drug target screening and immune factors screening.Currently,CRISPR libraries are mostly delivered by lentiviral vectors.However,because of the requirement of cumbersome preparation and demanding work conditions,security risk and immunogenicity,the application of viral delivery vectors is limited.Therefore,it is of great significance to develop an alternative delivery system for CRISPR libraries that could be used both in vitro and in vivo.Toward this end,here we have generated a mouse genome-wide CRISPR/Cas9 library based on the piggyBac transposon vectors.First,we verified that the mouse gene Tet1,Tet2,and Tet3 could be targeted successfully by using PB-CRISPR/Cas9 vectors.Then,we synthesized and cloned the sgRNA library into piggyBac transposon vectors to generate the PB-CRISPR/Cas9 library.The sgRNA library contains 130209 sgRNAs of which 1000 are nontargeting sgRNAs,and the other sgRNAs target 20611 mouse protein-coding genes and 1175 miRNAs with 4-6 sgRNAs per gene.To examine the integrity of the PB-CRISPR/Cas9 library,we performed a high-throughput sequencing analysis,and the result showed that 94.5%sgRNAs from the synthesized sgRNA library could be found,and distributed evenly on each chromosome.These results demonstrated that our PB-CRISPR/Cas9 library was constructed successfully.To investigate the efficiency and stability of in vitro screening by using PB-CRISPR/Cas9 library,we made a genome-wide screening for differentiation factors in mouse embryonic stem cells(ESCs).ESCs with the Nanog-GFP reporter were electro-transfected with library vectors.After two month culture in differentiation medium,we collected cells with normal proliferation.Results of immunofluorescent staining and quantitative PCR showed that pluripotency genes were expressed in these cells.We performed deep sequencing against the sgRNAs amplified from these cells,and identified genes related to stem cell self-renewal maintaining.Most genes found are related to differentiation,such as Tcf7l1,Csnk1a1,Socs3,and Flcn.These results indicated that the PB-CRISPR/Cas9 library can be used for efficient large-scale in vitro screening.To examine the feasibility of in vivo screening with PB-CRISPR/Cas9 library,we conducted a genome-wide screening for tumor suppressor genes in mouse livers.To accelerate the tumorigenesis,we used Cdkn2a knock-out and hNRASG12V overexpression vectors to sensitize the genetic background.The results showed that tumors could be obtained within two months.Next,high-pressure tail vein injection was performed to deliver PB-CRISPR/Cas9 library,Cdkn2a knock-out,and hNRASG12V overexpression vectors into ICR male mice.The mice were examined at 45-60 days postinjection,and liver tumors were detected in nine of 27 mice.All of the tumors were analyzed by hematoxylin-eosin staining and immunohistochemistry,and the results showed that most tumors were intrahepatic cholangiocarcinoma(ICC).In these tumors,genes mediating liver tumorigenesis were identified by deep sequencing analysis,most of them are tumor suppressor genes,including both known and unknown ones,such as Trp53,Cdkn2b,Parm1,and Sel1l2.The results suggest that PB-CRISPR/Cas9 library could be directly used to perform in vivo screening.In summary,we produced a PB-CRISPR/Cas9 library for genome-wide screening.We conducted both in vitro and in vivo screens and identified essential genes mediating cell differentiation and liver tumorigenesis,respectively.Our results demonstrate that the screening based on PB-CRISPR/Cas9 library is efficient and simple,and it is a good choice for genome-wide screening in the future.