Establishment Of An Efficient And Simple CRISPR/Cas9 Genome Editing System In Vivo And In Vitro And Its Application In The Study Of Sall4 In Medaka

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)is a powerful tool in genome manipulation.In mammals,CRISPR/Cas9 has been widely used for genome editing in cultured cells and whole organism.In fish,although CRISPR/Cas9 has been applied in gene function researchs in vivo in a variety of fish,its application in cultured cells still faces great challenges and limitations.Cultured cells are useful models for the study of virology,environmental toxicology,genetic breeding,resource conservation and molecular mechanism.Compared with in vivo,gene function researchs in fish cultured cells can not only save time and costs,minimize the influence of genetic heterogeneity,but also overcome the limitations such as the long spawning cycle of some fish species.However,there are still some shortcomings exist in reported CRISPR/Cas9 mediated gene manipulation in fish cultured cells such as low efficiency,high cost and complex operation.Therefore,an efficient,simple and economical CRISPR/Cas9 genome editing tool for fish cultured cells is urgently needed.On the other hand,spalt-like transcription factor 4(Sall4),a member of the spalt-like zinc finger transcription factor family,plays an important role in the proliferation and differentiation of mammalian spermatogonia,but its function in fish spermatogonia has not been reported.Based on these,an integrated plasmid based CRISPR/Cas9 system was eatablishmented in this study,different fish U6 promoters were used to drive sgRNA expression,CMV promoter was used to drive Cas9 expression and SV40 promoter was used to drive neomycin resistance gene expression.The gene editing efficiencies of the integrated plasmid pCas9-U6 sgRNA containing different fish U6 promoters were compared mutually with help of the enhanced green fluorescent protein(EGFP)reporter plasmid in medaka(Oryzias latipes)cultured cells.The knock-out and knock-in efficiencies in endogenous gene manipulations were evaluated in medaka cultured cells and embryos.On the other hand,this system was further applied to study the function of medaka Sall4(Olsall4).The SG3 single-cell clones with homozygous phase-shift mutation of Olsall4 were successfully obtained,combined with vivo-Morpholino antisense oligonucleotide mediated gene knockdown,it was found that Olsall4 knock-out or knock-down did not affect the proliferation and survival of SG3.The details are as follows:1 Establishment of an efficient and simple CRISPR/Cas9 genome editing system for medaka in vivo and in vitro1.1 Cas9-sgRNA co-expression plasmids and their gene editing efficienciesCas9-sgRNA co-expression plasmid pCas9-z U6 sgRNA containing zebrafish(Danio rerio)U6 promoter was constructed in previous study,U6 promoters from medaka and Nile tilapia(Oreochromis niloticus)were used to replace the zebrafish U6 promoter and named as pCas9-m U6 sgRNA and pCas9-Nt U6 sgRNA,respectively.Gene editing efficiencies of pCas9-U6 sgRNAs containing U6 promoters of medaka,Nile tilapia or zebrafish were compared in medaka SG3 and embryonic stem cell MES1 by using an EGFP reporter plasmid pGNtsf1.The results showed that the gene editing efficiency of pCas9-m U6 sgRNA was the highest,followed by pCas9-Nt U6 sgRNA and pCas9-z U6 sgRNA was the lowest.These results suggested that the relationship between U6 promoters and cultured cells is an important factor affecting the gene editing efficiency of pCas9-U6 sgRNA in fish cultured cells.1.2 Dosage optimization of pCas9-U6 sgRNA in medaka cultured cellsDifferent dosages(10,50,100,200,300,and 400 ng)of pCas9-m U6 sg Ntsf1 were co-transfected with 200 ng reporter plasmid pGNtsf1 in SG3 at 24-well cell culture plates.The results showed that with the increase of the dosage of pCas9-m U6 sg Ntsf1,the percentage of EGFP positive cells increased and peaked at the dosage of 300 ng.The results showed that 300 ng was the best transfection dosage.1.3 Endogenous gene knock-out mediated by pCas9-m U6 sgRNA in medaka cultured cellsTotal 8 sgRNAs targeting 4 endogenous genes were designed and the editing efficiency at each target site was tested.The results showed that the editing efficiency of6 targets was higher than 31% and the highest was 93.7% after G418 selection.Total 4potential off-target sites were detected,and no off-target was observed.These results indicated that pCas9-m U6 sgRNA can effectively knock out endogenous genes in medaka cultured cells.1.4 Gene knock-in mediated by pCas9-m U6 sgRNA in medaka cultured cellsIn addition,we designed and constructed the targeting vector and knock-out vector targeting medaka actb2 to detect the gene knock-in efficiency mediated by pCas9-m U6 sgRNA in medaka cultured cells.The results showed that puro-egfp was correctly inserted in front of the actb2 stop codon and was expressed.These results indicated that pCas9-m U6 sgRNA can effectively mediate exogenous gene knock-in at specific genome sites in medaka cultured cells.1.5 Gene knock-out and knock-in mediated by pCas9-m U6 sgRNA in medaka embryosIn addition,we also found that the pCas9-m U6 sgRNA system could mediate both endogenous gene knock-out and exogenous gene knock-in in medaka embryos by 1-cell embryo microinjection.2 Application of pCas9-U6 sgRNA in the study of Sall4 in medaka2.1 Isolation,identification,and expression pattern of medaka sall4The complete open reading frame c DNA of Olsall4 is 3192 bp,encoding 1063 amino acids.The protein structure,phylogenetic tree and gene syntenic analysis showed that Olsall4 is the homologous molecule of mammalian.By RT-PCR,Olsall4 was expressed in medaka embryos from 1-cell to gastrula stage,and was highly expressed in the testis and ovary in adult medaka.By in situ hybridization,our study showed that Olsall4 was expressed in 4-cell,16-cell and morula embryos;in adult testis,Olsall4 expression was restricted to spermatogonia;in adult ovary,Olsall4 expression was restricted to oogonia and all stages of oocytes.2.2 Olsall4 knock-out single cell clones generated with pCas9-m U6 sgRNApCas9-m U6 sgRNA targeting Olsall4(pCas9-m U6 sg Olsall4)was constructed and transfected into SG3.After G418 selection and limiting dilution,three SG3 single cell clones with homozygous frame shift mutation of Olsall4 were successfully obtained,named as Clone1,Clone2 and Clone3 respectively.2.3 The role of Olsall4 in proliferation and survival of SG3The cell proliferation of three SG3 single cell clones with homozygous frame shift mutation of Olsall4 was examined,and the results showed that the cell proliferation was not significantly different from that of wild type SG3.Knockdown of Olsall4 by vivoMorpholino did not affect the proliferation and survival of SG3.The acquisition of SG3 single cell clones with homozygous frame shift mutation of Olsall4 also confirmed that Olsall4 is not required for the survival of SG3.These results suggest that Olsall4 is not essential for the proliferation and survival of SG3,which is different from its role in mammalian spermatogonia.In summary,we have successfully established a novel plasmid-based Cas9-sgRNA co-expression system,which is simple,efficient and economical in medaka genome editing in vitro and in vivo.Furthermore,we constructed an EGFP reporter plasmid which can be used to quickly and intuitively evaluate the gene editing efficiency of different pCas9-U6 sgRNAs.Importantly,our study suggests that the genetic relationship between the U6 promoter and the cells is an important factor affecting the gene editing efficiency of pCas9-U6 sgRNAs in fish cultured cells.This study lays an important foundation for gene editing and gene function study in medaka,and provides a new insight for the study of gene editing in other fish species.