Mouse Genome-wide CRISPR/Cas9 Knockout Libraries Were Used To Screen Genes Related To Propofol Causing Neurotoxicity

Objective:Compared with other anesthetic drugs,propofol is widely used because of its rapid onset,rapid awakening,and few side effects(such as nausea and vomiting).In recent years,more and more studies have shown that propofol can have neurotoxic effects on developing animals.We do not fully understand the mechanism of propofol-induced neurotoxicity,and the efficient and rapid screening of propofol-induced neurotoxicity related genes will provide a preliminary theoretical basis for subsequent research on molecular mechanisms and signaling pathways,and provide a theoretical basis for new drug development and targeted clinical intervention.Methods:1.Mouse hippocampal nerve cells(HT22)were cultured with DMEM high-sugar complete medium containing propofol concentrations of 0μg/m L,20μg/m L,30μg/m L,40μg/m L,50μg/m L,60μg/m L,70μg/m L,80μg/m L,90μg/m L,and 100μg/m L for 24 h,48h,and 72 h,respectively,and the cell activity was detected by CCK8 experiment,and the appropriate propofol screening concentration was selected.2.Using mouse CRISPR/Cas9 whole genome knockout library plasmid for lentiviral packaging,HT22 cells(control MOI≤0.3)were infected after testing viral titers,and successfully infected cells were screened after puromycin resistance screening.The infected cells were divided into three groups,one group was directly frozen as the baseline group,and the other two groups were the experimental group and the control group,the experimental group was cultured in a propofol environment,the control group was cultured in a propofol-free environment,cultured for 7 days,the surviving cells were collected,and the whole genome DNA was extracted from each group,and after the accounting quality test was completed,high-throughput sequencing was performed to obtain the candidate genes corresponding to the enrichment of sg RNA.Through GO analysis and KEGG analysis of candidate genes,relevant biological processes,molecular functions and metabolic pathways were obtained.At the same time,PPI analysis was performed on the differential genes to find the key genes of propofol induced neurotoxicity.Results:1.Lentiviral packaging and lentiviral transfection of HT22 cells were completed using mouse CRISPR/Cas9 genome-wide knockout plasmid library.2.It was verified that propofol has a neurotoxic effect on nerve cells,and the toxic effect increases with the increase of propofol concentration.We cultured HT22 cells with DMEM high-sugar complete medium containing propofol concentrations of 0 μg/m L,20μg/m L,30 μg/m L,40 μg/m L,50 μg/m L,60 μg/m L,70 μg/m L,80 μg/m L,990 μg/m L,and 100 μg/m L,respectively,and found that the survival rate of HT22 cells decreased with the increase of propofol concentration in the medium and the extension of the culture time.3.After the sequencing is completed,we perform quality control on the sequencing data,and the sequencing quality analysis shows that the effective data rate: >90%;Comparison rate: >90%;Zerosg RNA: < 100;Zero genes: <10;read count distribution of g RNA:Poisson distribution;Gene read count box plot: can be concentrated into boxes and the height of the box is narrow;Giniindex:<0.1;The representative library is of high quality and can be analyzed for the next step.4.Through the comparison of differences between samples,it was found that there were2867 positive screening genes and 2193 negative screening genes,and the top ten genes with significant differences in positive and negative screening were visualized and displayed,and the positive screening genes included Pcdhgc5,Rps 29,Rps 21,Rps3,Atp5 k,Rngtt,Uba52,Pcdhga12,Pcdhga5,Mup1,etc.Negative screening genes include Bcl2l1,Vdac2,Pfkp,Slc2a1,Pgd,Tmsb4 x,Krtap2-4,Aldoa,Cept1,etc.5.The differential genes screened out were enriched by GO and KEGG.The KEGG enrichment pathway of significantly different genes mainly included pentose phosphate pathway,aminoacyl-t RNA biosynthesis,nucleotide excision repair,linoleic acid metabolism,homologous recombination,amino sugar and nucleotide sugar metabolism and other basic metabolic pathways.Significantly different gene GO enrichment pathways mainly include: 1.Biological processes: glycolysis,assembly of small ribosomal subunits,transmission of mitochondrial electronic chains,assembly of mitochondrial respiratory chain complexes,DNA-dependent DNA replication,protein translation,etc.2.Cell components: cytoplasmic exosomes(ribonuclease complexes),cytosolic small ribosomal subunits,transcriptional active chromatin,polyrisomes,Mll1 complex,eukaryotic translation initiation factor 3 complex,mitochondrial small ribosomal subunits,etc.3.Molecular function: small ribosomal subunit r RNA binding,translation initiation factor activity,r RNA binding,transcription factor complex binding,translation initiation factor binding,ATPase activity,transmembrane transporter activity,etc.6.PPI analysis was performed on differential genes with diff values of >0.25,and 12 key genes leading to neurotoxicity were screened for propofol,Uba52,Rps13,Imp3,Rpl7,Rps3,GAPDH,Mrpl22,Atp5a1,Rps29,Rps28,Polr3 a,Rps27.Conclusion:1.Under the intervention of propofol,the cell activity of mouse hippocampal neurons showed a dose-dependent downward trend.2.Through high-throughput sequencing and bioinformation analysis,the differential genes and key genes that can cause neurotoxicity in propofol were screened,as well as related metabolic pathways and biological processes.Among them,we found that mitochondrial and ribosomal related biological processes such as mitochondrial electron chain transmission,mitochondrial respiratory chain complex assembly,mitochondrial transmembrane transport,ribosome biogenesis,protein translation,etc.were significantly enriched,representing that mitochondria and ribosomes play an important role in the process of propofol causing neurotoxicity.