Study On Crispr Technology Suitable For Gene Function Research Of Stylonychia

S.lemnae have been studied thoroughly as the type species of the genus Stylonychia.It is an important organism for studying cell biology and molecular biological mechanisms.The genome determination of S.lemnae has been completed which makes it possible to study the gene function of S.lemnae.Gene knockout is the most effective way to study gene function.However,there is no reference for efficient and accurate gene knockout to verify gene function in protozoa,especially in S.lemnae.CRISPR/Cas9 system is the fastest developing genome editing technology in recent years.It can achieve gene editing at a fixed point.It is widely favored by researchers because of its high efficiency and flexibility.The establishment of CRISPR/Cas9 system suitable for S.lemnae can effectively promote the study of gene function of S.lemnae.This study began with studying the codon usage bias of S.lemnae,then a CRISPR/Cas9 gene editing system was established in S.lemnae,and the function of AdSS gene was tested in order to establish a CRISPR technology platform suitable for the study of the gene function of S.lemnae.The main results are as follows:By calculating the GC content of codon 3 of S.lemnae,it was found that codon 3was rich in A and U.PR2-plot mapping of the genome of S.lemnae duckweed showed that the frequency of the third base T was higher than that of A,and that of C was higher than that of G.Combined with neutral mapping analysis,ENC-plot mapping analysis and PCA analysis,it was found that codon preference of S.lemnae genome was greatly influenced by mutation and natural selection,but also by other factors.Codon preference was the result of multiple factors.Finally,24 codons,such as AGA,GGA and UUA,are determined to be the best codons by high expression superior codon method.A gene AdSS annotated with adenylate succinate synthase was cloned from S.lemnae by PCR amplification.The sequence alignment of nucleic acid and protein showed that AdSS gene of S.lemnae was highly homologous to Paramecium digitatum,Cucurbita multifilaria and Tetrahymena thermophila.The vector for locating AdSSgene was constructed by simultaneous digestion of DNA and pEGFP-N1 vector.Because it is mainly located in the cytoplasmic ribosome.According to the analysis of codon usage bias of S.lemnae,Cas9 protein in the optimal codon modification vector was obtained,and the successful mutant plasmid was screened for transfection.At the same time,several sgRNA targeting sites were designed for AdSS gene in S.lemnae.And one sgRNA was successfully obtained to act on the target gene and produced large deletions.When the AdSS gene was knocked out,the movement of the cells was obviously slow or even completely immobile,the growth and development of the cells were also affected,and a small number of the cells were deformed.In order to detect knockout efficiency,real-time fluorescence quantitative PCR was used to detect mutants,which showed that the CRISPR/Cas9 system could efficiently and specifically implement genome editing in S.lemnae.To sum up,this study screened and validated the optimal codon of S.lemnae,optimized the knockout vector,and successfully used CRISPR/Cas9 system to achieve the specific knockout of AdSS gene of S.lemnae.It proved that this system could be effectively used for genome editing of S.lemnae.The obtained stable mutation cell cloning and constructed CRISPR targeting vector.The research has laid a solid foundation for subsequent study of the gene function of S.lemnae.