Systematic Studies Of The Circadian Clock Genes In Mouse Regulation On Temperature Compensation And Cell Cycleusing CRISPR Cas9 Genetic Editing Tools

The mouse circadian clock is regulated by circadian genes,and the circadian genes are capable of producing a self-sustained,autonomous oscillating rhythm of-24 hours.A major regulatory pathway present in the circadian rhythm is the formation of heterodimers by the core circadian components BMAL1/CLOCK,which binds to the E-box region of the transcriptional repressor PER and CRY promoters,and then activates transcription of per and cry.When a certain concentration of PER and CRY is reached,PER and CRY form a heterodimer which enters the nucleus to detach BMAL1/CLOCK from the E-box,thus inhibiting its activation.In addition,there are many genes involved in the regulation of the circadian clock,such as transcription factor genes:mciart,mdbp,mnfil3,mdecl,mdec2,mnpas2;nuclear receptor family genes:mrora,mrorb,mrorc and mnrldl,mnrld2,which regulate mbmal1 transcription by binding to the RORE binding site of mbmall gene as positive and negative regulators,respectively;E3 ligase genes that mediate ubiquitination of PER and CRY proteins:mbeta-trcp and mfbx1211,mffbxl3;phosphokinase family genes that mediate PER and CRY phosphorylation:mcsnk1d,mcsnk1e,and mtimeless and mmyc,which are currently not studied well in circadian rhythms at the cellular level.The purpose of this thesis is to use the CRISPR genetic editing method to systematically study the regulation of the circadian rhythm length,temperature compensation and cell cycle by these 25 genes.Targetting these 25 genes as research objects,22 individual cell lines were succesfully edited in mouse fibroblasts by CRISPR cas9 gene editing system,and no off-target phenomenon was found after off-target analysis.Through the Lumicycle Reporter experiment,the period changes of the obtained gene-edited cell lines were compared with those of the gene knockouts mouse models reported in the literature,and the trends between them were similar.The calculation of the temperature compensation coefficient Q10 at 32.5 ?and 37 ? lead us to a conclusion that the editing of most single gene does not have a great impact on the temperature compensation,but the mcsnkle and mbeta-trcp Q10 is less than 0.8,these two genes may be related to the maintenance of temperature compensation;the mbmall,mcryl,mperl,mper3,mfbx13 genes have a rhythmic cycle that decreases with increasing temperature,while others are reversed.It shows that the temperature compensation can be achieved by the period-increasing and period-decreasing processes balance,and temperature compensation is a complex gene regulation system.In the process of studying the cell cycle regulation of these genes,the cell growth curve was first measured by MTT colorimetry.Then the growth curve was fitted by the two-split growth curve function,and the obtained celldoubling time was calculated.The strain mclock-F12.mcry1-D4,mper1-B7.mper2-A3.mper3-D4,mnrld2-F4,mcsnkle-B5 has significant longer in cell proliferation rates,other strains had little change on the cell doubling time.We further carried out flow cytometry analysis with these strains,we found that mcry1-D4,mperl-B7,mper1-C1,mfbxl3-A9,mfbxl21-C11,mrora-D2,mdec2-E8 and mnfi13-B12 cell lines were arrested in G1 phase;the mbmal-D8 and mcry2-H3 cell lines were arrested in G2/M phase;mcsnkld-D2 cell lines were arrested in G2/M phase.In addition,in terms of cell strain;mcsnkle-B5,the S phase cells were significantly lower than the wild type,and the mclock-F12,mcry1-C9,mbeta-btrc-C9,mnpas2-B8,mdbp-F4,mnr1d2-F4 cell line has significantly change G2 phase.The mnpas2-B8 cell line even could contain doubled chromosomes.These results indicate that a major aspect ofcircadian regulation of the cell cycle is through cell cycle checkpoints.Our study targets to edit 25 ciacadian-related genes by CRISPR cas9 gene editing method.On one hand,we can systematically study the effects ofthese genes editing on biological rhythmsand temperature co,mpensation;on the other hand,through the study of the length and phase distribution of cell cycle after editing of these genes,the role of circadian genes in cell cycle regulation was systematically studied.