Optimization Of CRISPR Technology And Modification Of PAM Recognition Sites Of Cas9 Enzyme

Genome-editing is a powerful approach to study the functions and applications of targeted gene in various organisms including mammals and flora.Recently,the cluster regularly interspaced short palindromic repeat and its associated protein(CRISPR/Cas)systems have been rapidly developed particularly the CRISPR type II named CRISPR/Cas9.Cas9 can be guided to the sequence-specific genome region by a short complementary RNA,then cleavages the targeted DNA to generate the double-strand breaks(DSBs)through its nuclease domains.During the DNA repair,the NHEJ may cause indels and HDR can result in site-specific mutation with the recombinant template,which would generate frame-shift mutation and disrupt the gene functions.Compared with ZFNs(zinc-finger nucleases)and TALENs(transcription activator-like effectors nucleases),the CRISPR/Cas9 system is less time-consuming,more efficient and easier to implement technology,hence it's commonly utilized in model plants and crops.1.Based on the plant-codon optimized method,we optimized the Sp Cas9,increasing the GC contents to about 49%,which let the Cas9-codom optimized express much more stable in vitro.We constructed the CRISPR/Cas9 binary expression vector using Zm Ubi as the promoter of Cas9,and the TaU6 as the promoter of sgRNA scaffold,and selected the TaAGO4 a as the targeted gene to test its function.We transformed the CRISPR/Cas9-TaAGO4a/sgRNA1 into the wheat protoplasts and obtained various mutated types,including-6bp,-1bp,+1bp,+84bp and so on.The mutated ratio was about7.00%.Then we transformed it into wheat young embryos,and obtained 36 transplants.We also tested one plant,named L19,deleted 7bp in TaAGO4a-3A.The mutated ratio is about 2.78%.2.In order to expand the use of CRISPR/Cas9 technology,we have modified the PAM site.The classic CRISPR/Cas9 system was restricted by its PAM: NGG,which desreased its applications in plant research.Utilizing the bioinformatics method,we analyzed the CDS of wheat whole genome regions.We found that the number of PAM: NGA is much more than PAM: NGG,increasing about 24.78%.In rice,we also discoveried that the number of PAM: NGA is more than PAM: NGG,arising about 12.34%.Therefore,we can modify the Cas9 protein to change its PAM from NGG to NGA,which can make it more powerful than previous.We utilized the PCR site-directed mutation technique to remould the1135 D to 1135 V,1335R to 1335 Q and 1337 T to 1337 R respectively.We named it as Cas9-VQR,and constructed binary expression vector of CRISPR/Cas9-VQR,which contained the ZmUbi to express the Cas9-VQR,and Os U6 to express the sgRNA scaffold.We selected OsPDS(the phytoene desaturase)as the targeted genes to verify its function.Using the enzyme activity test in vitro,we found the modified Cas9-VQR can cut the targeted sites,the mutated ratio was from 5% to 70%.Then,we tansformed them into rice young embryos respectively.Throught sequcing the targeted fragment,we tested variour mutated types containing deleted 19 bp,inserted 28 bp and so on.The mutated ratio was from 27.50% to70.50%,and the average mutated ratio is 46.23%.3.We did some modification and attempt of the CRISPR/Cpf1 Technology.Compared with CRISPR/Cas9,fn Cpf1 recongized the PAM: 5'TTN,and generated the staggered cut.Meanwhile,the sgRNA scaffold was much shorter than previous,ones made it more convenient to generate the Cpf1-sgRNA complex in vivo.Therefore,we optimized the Lb Cpf1 codons based on plant-codon preference and constructed the binary expression vector of LbCpf1.In order to test its function,we selected the OsPDS as a targeted gene.We transformed them into rice young embryos respectively.We obtained albino plants with white-green leaf or sheath phenotypes.Unfortunatly,we didn't detect the deletion or insertion in the targeted regions.Through qPCR,we tested the relative expression level of OsPDS in transplants,and found that the expression of OsPDS decreased from various levels.We hpothesised that the CRISPR/Cpf1 can bind the targeted sites without cleveage,but may prevent gene transcription.In summary,we made some modification on CRISPR/Cas9 and CRISPR/Cpf1 technology,and verified in rice and wheat.Our work shows that We extend the scope of the application of CRISPR Technology and may contribute to the breeding and improvement of crops.