| The emergence of genome editing technology is the inevitable result of the gradual completion of genome sequencing of various species.In this process of exploration and decoding,three generations of artificial nuclease gene editing technology have been progressively updated.From ZFNs,TALEN,to CRISPR/Cas system,whether it is ordinary cell lines or living individuals,they have played a great role,for instant,in promoting the study of gene functions on the field of scientific researches,in production of transgenic crops and animals on the field of biotechnology application.However,the complex context of eukaryotic genome,the diversity of intracellular environment,and the potential harm caused by DSBs are obstacles that are difficult to be overcome in genome editing.In order to achieve the optimal genome modification as far as possible,the best system composition,transmission mode and detection method by constantly optimizing,maybe which is the second best but hopefully and most effectively.This study extends to detecting the activity and editing efficiency of the nuclease in the genome editing system,the main purpose of this test is constructing a convenient Indels detection system for laboratory daily use,and real reflecting the working efficiency of CRISPR/Cas9 editing system(not specific)simply and effectively.And further provide the data support for the optimization of the genome editing system,promote the gene engineering technology to achieve the ideal editing on interest genes,and even forward promote the transgenic production and the treatment of human genome genetic diseases.This system mainly relies on the generation of various Indels mutations at the cleavage sites by NHEJ repair mediated by CRISPR/Cas9 gene editing system,with the help of the mature prokaryotic L-arabinose induced expression system,by screening the mutations yield to on-frameshift of m Fab I marker gene to revealing the Indels frequency,in the form of the proportion of screened clones to present the results.Due to the characteristics of the construction system,it can realize more accurate Indels detection on different cell types and different gene loci.The main results of this experiment are as follows:1.Successfully construct detection system and achieve system function verificationBased on the preliminary verification of Amp R gene as the screening marker gene to detect Indels mutation,we determined that it was feasible to use the inducible expression system in bacteria as the basis for Indels mutation detection.In the wild type 293 T genome(CCR5 gene,EMX1 gene,and HPRT1 gene),we identified the feasibility of Indels mutation detection basing on m Fab I gene expression induced by L-arabinose.Under the conditions of on-frame or frame recovery,bacteria Fab I(G93V)protein normally expressed,generated Triclosan-resistance,and grow up on the screening culture medium containing Triclosan with L-arabinose.2.Determine the feasibility and practicality of detecting systemAfter the Indels detection system been respectively applied on three group genomes had induced by CRISPR/Cas9 system,which including screened and unscreened genome at different target genomic locus(CCR5 gene,EMX1 gene,and HPRT1 gene),genome of different cell lines(293T,A375,U2OS)at the same locus(CCR5 genes),we determined that the minimum detectable frequency of Indels could not less than 1% and modified by clones sequencing.Compared with the results of amplicon sequencing(NGS)analysis,the distribution of Indels type and frequency showed a good consistency,which basically accomplished the quantitative and semi-qualitative revelation of Indels and could effectively evaluate the activity and efficiency of nucleases.To sum up,this study provided a convenient and effective detection system for the filed of genome editing and mutations detection,which provided strong data support for the evaluation of genome editing efficiency and nuclease working efficiency,and laid a foundation and provided forward momentum for the optimization and application of gene editing system... |
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