Inhibition Of Intracellular Non-targeted DNA Cleavage Digestion By Cas9 And RNP Complexes And Characterization Of CRISPR/Cas9 Off-target

Since the CRISPR/Cas9 system has been used in genome editing,it has accelerated the research process in the fields of biology,agriculture,and medicine.However,there are several problems with this technology,a major one being its ability to induce mutations in non-targeted genes.Currently,it is thought that the main cause of mutations in non-target genes is non-specific binding and cleavage.Increasingly,studies have shown that off-target sites predicted by off-target site prediction software are mostly unmutated,but instead unpredicted sites are mutated and in an irregular manner.In addition the off-target types are mostly mutations of single nucleotides(SNVs)rather than insertions and deletions(Indels).This indicates that the causes of non-target induction in CRISPR/Cas9 systems are not limited to non-specific binding and cleavage.Therefore,researching the effects of CRISPR on targeted and non-targeted nucleic acid sequences in a more proximate intracellular environment will help analyze the reasons for CRISPR non-target.In this study,the Nicotiana benthamiana leaf cell-free system was used to simulate the vivo environment to study the effect of CRISPR complex on DNA.we constructed protein expression vectors to express Cas9,nCas9,dCas9 proteins,RNP(Ribonucleoprotein,Cas9+sg RNA),nRNP(nCas9+sg RNA),and dRNP(dCas9+sg RNA)recombinant proteins in tobacco,detecting the digestion and cleavage activity of tobacco leaf cell extracts against targeted and non-targeted DNA fragments;Meanwhile,We analyzed the whole genome sequencing data of Cas9-expressing mice and nCas9-expressing rice.The results showed that the CRISPR component inhibits non-targeted cutting and digestion,thereby inhibiting the repair of mutation sites in the cell and promoting the generation of non-targeted mutations.The main results are as follows:1.Nicotiana benthamiana leaf cell extracts can digest DNA fragments with stable digestion ability.In the experiment,a series of linear DNA fragments of different lengths and supercoiled plasmid DNA were designed,and the digestibility of tobacco cell extracts was determined through a 2-hour time gradient reaction.We compared the digestion results of different length DNA fragments and the same length but different structural state DNA in leaf cell extracts.The results show that Nicotiana benthamiana leaf cell extracts can digest fragments of different lengths and have stable enzyme kinetic activity.2.The digestibility of tobacco cell extracts was inhibited by Cas9 and its variants.In the experiment,the green fluorescent gene GFP was connected to the Cas9 protein to construct the p KSE401-GFP vector.The expression of the Cas9 protein can be verified by observing the fluorescence phenotype;at the same time,two variants of Cas9,nCas9(Nickase Cas9),and dCas9(DeadCas9)were constructed by site-directed mutagenesis.Nicotiana benthamiana was infected by the Agrobacterium infection method,and cell extracts were prepared for in vitro DNA digestion experiments.The results showed that cell extracts expressing Cas9,nCas9,and dCas9 can inhibit DNA digestion.3.Reseaching on the specificity ofRNP complexes in cell extracts.We selected two suitable target sites,constructed p KSE401-sg PDS and p KSE401-sg GFP vectors,verified the editing effect of the target sites,and showed the effectiveness of theRNP complex expressed by the vector in tobacco,and laid the foundation for the expression of exogenousRNP in Nicotiana benthamiana.We selected exogenous targeting genes GFP and Lac Z,constructed the corresponding expression vectors ofRNP,nRNP,dRNP,and transiently infected Nicotiana benthamiana by Agrobacterium to prepare cell extracts for in vitro experiments.The results showed that cell extracts expressing targetedRNP accelerate the digestion of target DNA;cell extracts expressing nRNP and dRNP inhibit DNA digestion.In the experiment,the specificity ofRNP complexes in cell extracts was explored by constructing double base mutations and deletios target-GFP.The results showed thatRNP cell extracts inhibited non-targeted digestion.4.This study analyzed the whole genome sequencing data of Cas9-expressing mice and-expressing nCas9 rice.It was found that the off-target mutations induced by CRISPR were inconsistent with the cleavage principle,off-target mutations were random,and off-target mutations were biased towards PAM sites.The above results indicated that non-target mutations is not due to cleavage of Cas9,but the inhibition of repair of non-targeted sites caused by the binding of Cas9 to the PAM site..In this study,the cell-free system was used to detect the targeted and non-targeted digestion and cleavage effects of Cas9 andRNP complexes,indicating that Cas9 and its variants,nRNP and dRNP have a certain inhibitory effect on the digestion and cleavage of DNA from cell extracts.Combined with CRISPR-induced mouse and rice genome-wide sequencing data analysis,it is shown that the CRISPR system induces off-target effects because Cas9 recognition and binding of non-target sites inhibits genomic DNA repair,thereby promoting spontaneous mutations in organisms.