| RNA interference(RNAi)is a ubiquitous post-transcriptional gene expression regulation mechanism in eukaryotes,which has been developed as a powerful reverse genetics tool,showing great potential in many fields,such as gene function analysis,pest control,gene therapy and so on.However,dsRNA has obvious off-target effects.First,exogenous dsRNA can produce specific off-target effects related to dsRNA sequences by silencing non-target homologous genes.Second,at higher doses,dsRNA will also produce non-specific off-target effects that are not related to dsRNA sequences through biochemical pathways such as immune and metabolic feedback.Off-target effect will lead to wrong conclusion of gene function analysis in scientific research,that is,the functional effect of non-target gene silenced is mistaken for the function of target gene,and will also produce unexpected biological effect in the field of application.Especially in the field of medical research,off-target effect induced by nucleic acid drugs will lead to a variety of side effects,which seriously hinder the development,research and application of drugs based on RNAi.In the field of agricultural pest control,if the dsRNA released to agricultural fields induce off-target effect,it will not only cause unpredictable off-target damage to environmental organisms,but also probably endanger human health.In this paper,we focus on the specific off-target effects related to dsRNA sequence,systematically explore the factors that affect off-target effect from the view of gene expression level,sequence identity and base sequence matching characteristics,and summarize the guidelines of off-target effect mediated by dsRNA sequence,and establish a prediction and evaluation system of off-target effect mediated by dsRNA.The results are significant for both theoretical studies and practical applications of RNAi,and should promot the extensive research and application of RNAi measures for pest control.1 Optimization of experimental conditions for RNAiIn order to eliminate experimental interference and accurately identify the key factors that affect RNAi efficiency,this study first optimized the experimental conditions of RNAi.Firstly,we compared six genes(Drip,CYP4G7,CYP6BK13,AANAT1,CYP6BQ9 and CYP6BQ10)in Tribolium castaneum,in which each gene was selected at 2～3 different positions to synthesize a 100 bp dsRNA as a template,using RNAi experiment to detecte the RNAi response of different gene targets to dsRNA.The results showed that there was no significant difference in RNAi effect among different positions of the same genes except for Drip.For Drip,there was no significant difference between position 1 and 2 as well as 2 and 3,only position 1 and 3 showed statistically significant difference,but the RNAi efficiency difference was only 11.9%,which did not affect the results of efficient RNAi effect of all three positions.Therefore,RNAi targeting at different positions of the same gene generally does not significantly affect the RNAi efficiency of dsRNA.Secondly,we synthesized 100 bp and 206 bp dsCYP6BQ6 to silence CYP6BQ6,and found that there was no significant difference in the RNAi efficiency of the two dsRNA,indicating that 100 bp dsRNA can produce efficient RNAi effects.Thirdly,we used dsRNA to treat the larvae of T.castaneum,and detected the RNAi efficiency changes of three different genes at different times after treatment,then found that the RNAi efficiency of target genes and non-target genes tended to be stable at 36 h after treatment,which indicated that the proper time of RNAi efficiency detection might be 36 h after dsRNA treatment.Fourthly,five genes with different expression levels were selected for RNAi test.The RNAi efficiency was detected by ddPCR and Q-PCR,respectively from the perspective of transcription molecular number and relative expression level.The results showed that the RNAi efficiency of the five genes measured by ddPCR was 96.5%,94.0%,83.9%,22.6%and 1.6%,respectively,while the RNAi efficiency measured by Q-PCR was 98.8%,95.3%,81.3%,21.9%and 0%,indicating that the two methods were basically the same(r=99.99%).Therefore,the relatively cheap Q-PCR method is reliable for large-scale detection of RNAi.This study preliminarily indicated that gene expression may be one of the key factors affecting RNAi efficiency.Fifthly,we tested the RNAi efficiency of dsRNA dose on different genes with different expression levels.The results showed that the response of different genes to the injection of 20～2000 ng dsRNA per insect was consistent.For the sensitive genes with high expression,0.018 ng of dsRNA can produce significant RNAi effect,while for the insensitive genes with low expression,even if the treatment dose is as high as 2000 ng per insect,it will not produce significant RNAi effect.Therefore,the injection of 200 ng dsRNA per insect can ensure the results of RNAi experiment with T.castaneum.At the same time,this experiment shows again that gene expression is one of the key factors affecting RNAi efficiency.2 Effect of gene expression level on RNAi efficiencyThis study focuses on analyzing the key factors affecting RNAi sensitivity of different genes in the same species.Firstly,we measured the relative expression levels of 90 genes in the untreated 5th instar larvae of T.castaneum by fluorescent quantitative PCR.At the same time,the same batch of test insects were subjected to RNAi tests in parallel to detect the sensitivity of these genes to their corresponding dsRNAs;and the correlation between the expression levels of these genes and RNAi sensitivity was analyzed.The results showed that the genes with more than 115.3 expression level were extremely sensitive to dsRNA,and the silencing efficiency was more than 80%,with an average of 89.1%,while the genes with expression levels between 8.2 and 115.3 showed fluctuating sensitivity to dsRNA,and the corresponding average gene silencing efficiency decrease to 63.8%.When the gene expression level was less than 8.2,the average silencing efficiency was 12.8%,and most genes were insensitive to dsRNA,and expression level of some genes were even up-regulated.Through statistical analysis,we found that the correlation coefficient between gene expression level and silencing efficiency was 0.8036 in T.castaneum,which was significantly positive correlation.Then,we proved this positive correlation between gene expression level and silencing efficiency by using L.migratoria and Drosophila S2 cells,and the correlation coefficients were calculated as 0.7255 and 0.9505,respectively.In order to eliminate the interference of other factors between genes,we selected three genes(CPRI8,Toll6 and Bib)whose expression level changed significantly during metamorphosis,and tested their expression levels and RNAi sensitivity in the larval and pupal stages,respectively.The results showed that when the test insects developed from larvae to pupa,the expression level of these three genes increased by 4.5,7.2 and 1.9 times,respectively,and their corresponding RNAi efficiencies increased from 36.4%,51.9%and 67.5%to 97.6%,76.3%,and 81.1%,respectively.In addition,the interference experiment of the same gene in different tissues showed that the higher the gene expression in a specific tissue,the greater the RNAi efficiency.All the results above indicate that expression level is a key factor in determining RNAi sensitivity of different genes in the same insect.3 The effect of sequence identity between dsRNA and target gene on off-target effectFirstly,we use a dsRNA synthesized with a specific gene to treat insects and detect the RNAi effect of the same dsRNA on different genes.The 5th instar larvae were injected by 100 bp dsCYP6BQ6,and then,the expression changes of 54 homologous genes(the identity to CYP6BQ6≥ 46%)were detected.The results showed that 9 genes with high sequence identity had significant off-target silencing effect,while none of the genes with sequence identity less than 68%had off-target silencing effect,indicating that the silenced genes’ sequence identity were highly similar with dsRNA sequence.Secondly,in order to rule out the influence of different gene expression levels,we randomly mutated the bases of target gene by artificial random mutation method,and synthesized a series of dsRNA with different similarity to silence the same gene.With five different target genes for replications,we found that the silencing efficiency of the mutated dsRNA to target gene gradually reduced along with the decrease of sequence similarity.When the sequence identity dropped to 80%or less,the dsRNA began losing the silencing ability.In addition,for CYP4G7 with high gene expression(600.1),silencing efficiency of the mutant dsRNA with identity>90%is almost the same as that of specific dsCYP4G7,and the knockdown efficiency was more than 90%.When the identity of dsRNA/gene decreased to 80%,the silencing efficiency dropped sharply to 0.For Drip,AANAT1,CYP6BK13 and CYP6BQ6,with lower expression levels(54-272),once the identity of dsRNA/gene decreased,the silencing efficiency of the corresponding mutated dsRNA to these four genes also decreased gradually,and when the dsRNA/gene identity decreased to 80%,the silencing efficiency decreased to 0.All these results demonstrated that the critical sequence identity of off-target effect is 80%.The higher the identity of dsRNA/gene is,the higher the probability of off-target effect is.The high expression of target gene can improve the sliencing efficiency,but can not change the critical similarity of dsRNA.4 RNAi off-target effect and the perfectly matching sequence of dsRNAIn the dsRNA-mediated off-target effects with different series identities,we found some special cases that though the whole dsRNA/gene similarity is relatively low,dsRNAs with longer perfectly matching sequences can also initiate off-target RNAi.In order to investigate the number of bases of the shortest perfectly matching fragments contained in the dsRNA that can initiate the silencing effect,CYP4Q7,which is extremely sensitive to dsRNA,was selected as the target gene.We prepared the templets by replacing EGFP gene sequence with 7-23 nt specific sequence fragments matching CYP4Q7and synthesize a series of 100 bp chimeric dsRNAs containing a perfectly matching fragments of different lengths to conduct RNAi experiments.The results showed that CYP4Q7 can not be silenced when the chimeric dsRNA contained only 7 to 14 nt matching sequence,but when the matching sequence length increases to 15 nt,chimeric dsRNA begins to have silencing ability,but the silencing efficiency is only about 20%.When the length of embedded matching sequence is between 15-18 nt,the silencing efficiency increases rapidly from 20%to 90%.When the chimeric dsRNA contains more than 18 nt matching sequences,all the initiated silencing efficiency can reach 90%,which is almost equal to that of 100 bp whole matching dsCYP4Q7.Then,CYP6BK13 with low expression was emplored as target gene to repeat this experiment.We found that the silencing efficiency of CYP6BK13 was about 20%when the perfectly matching sequence length in the chimeric dsRNA increased to 16 nt.When the length increased from 16 to 20 nt,the silencing efficiency of chimeric dsRNA gradually increased from 20%to 80%.When the length was more than 20 nt,the silencing efficiency of chimeric dsRNA tended to be stable.To sum up,for the sensitive genes,as long as containing a perfectly matching sequence of more than 15 nt,the chimeric dsRNA can initiate off-target RNAi.For the genes with lower sensitivities,the length of critical perfectly matching sequence will be longer.5 Off-target RNAi and the imperfectly matching sequenceof dsRNAIn the RNAi experiments mediated by dsRNA with different series identity,we also found that the dsRNAs without more than 15 nt continuously matching sequences could initiate off-target RNAi,but containing longer imperfectly matching sequences with single ortwo adjacent mismatchs.In order to clarify the basic characteristics of these sequences,we first used a series of evenly mutated dsRNA to conduct RNAi experiments,looking for the fewest contiguous matching bases connected by single and double mismach that can enable dsRNA to initiate RNAi.For connection of the single-base mismatch,we selected CYP4Q7,Drip,AANAT1 and CYP6BK13 as the target genes for repeated experiments.The results showed that for the highly expressed gene CYP4Q7,when the matching interval sequence length between single-base mismatch increased from 3 nt to 5 nt,dsCYP4Q7-5-1 began to appear significant silencing effect.For the genes Drip,AANAT1 and CYP6BK13 with middle expression level,when the sequence length increased to 6 nt,the significant silencing effect appeared.Using CYP4Q7 which is extremely sensitive to dsRNA as the target,we continue to screen the shortest matching interval sequence connected by double-bases mismatch.The experimental results show that when the length of the interval sequence increases from 3 nt to 8 nt,dsCYP4Q7-8-2 begins to appear silencing ability.When 18,21,24,and 50 nt of dsCYP4Q7-5-1 or dsCYP4Q7-8-2 were inserted into the EGFP sequence to synthesize a new dsRNA to silence CYP4Q7,the results showed that all new dsRNAs cannot silence CYP4Q7,which fully confirmed that a single-base mismatch connected 5 contiguous matching bases and two adjacent mismatched bases connected 8 contiguous matching bases was the critical imperfectly matching sequence for effective dsRNA.Since it has been proved that the shortest fragment of effective perfectly(contiguously)matching sequence is 15 nt,the naturally occurring effective imperfectly matching sequences should consist of 5-14 nt contiguous matching bases linked by a single mismatched base and 8-14 nt contiguous matching bases linked by two adjacent mismatched bases.Since the matching degree or identity of these sequences is higher than that of the critical sequence,it is speculated that the length of such sequences required for effective dsRNA will be shortened.In this study,we confirmed this speculation by comparing and analyzing the longest effective imperfectly matching sequences contained in effective and ineffective dsRNA,and found that randomly generated dsRNA containing effective imperfectly matching sequences≥ 26 nt could lead to off-target effect,and shortening the length of effective imperfectly matching sequences would make the off-target effect unstable and do not produce off-target effect until the imperfectly matching sequence<19 nt.6 Verification of the rules for off-target effect mediated by dsRNA and risk assessmentConsidering the gene expression level,sequence similarity,the shortest effective perfectly matchingand imperfectly matching sequence,we reveal the guidelines for dsRNA special off-target effect:1.non-target genes should be sensitive to dsRNA;2.The sequence identity between dsRNA and non-target gene is more than 80%;3.The length of the shortest perfectly matching fragment between dsRNA and non-target gene is≥15 nt;4.The length of effective imperfectly matching sequence between dsRNA and non-target gene is≥26 nt or at least between "warning zone"(19-26 nt),and theoretically,if condition 1 carrys one of 1,2 and 3 at the same time,dsRNA will have the risk of off-target effect on non-target genes.In this study,we proved these guidelines by predicting and testing the off-target effect of dsCsEF1 on six insects including Tribolium castaneum,Locusta migratoria,Bombyx mori,Spodoptera litura,Ostrinia furnacalis,Helicoverpa armigera as well as Drosophila S2 cells.According to the expression level and sequence characteristics of these homologous genes in different insects,we speculate that off-target RNAi effect will happen,except for lepidoptera insects which are not sensitive to RNAi.The experimental results were consistent with the prediction:the silencing efficiency of dsCsEFl against T.castaneum EF1 was as high as 90%,the EF1 of Drosophila S2 cell and L.migratoria was knockdown by 60%and 45%,respectively,but there was no obvious silencing effect on lepidoptera insects EF1,and even the silencing efficiency to its own target gene,Chilo suppressalis EFl,was only 20%.In addition,we also evaluated the off-target effect of dsDvSnf7 expressed in transgenic maize to control Diabrotica virgifera.The 240 bp D.virgifera Snf7 gene sequence was obtained by gene synthesis.The sequence identity with the corresponding region of T.castaneum Snf7 was 72%(<80%),but the matching sequence contained an effective imperfectly matching sequence with the length of 20 nt,which had just entered the off-target warning zone(19-26 nt).Therefore,we predict that even if the gene is sensitive to RNAi,it is difficult to achieve RNAi off-target effect.The experimental results showed that the lethality rate of dsTcSnf7 to the T.castaneum larvae was 100%,indicating that the Snf7 was sensitive to RNAi,but the silencing efficiency of dsDvSnf7 to T.castaneum Snf7 was only 24.2%,and there was no phenotypic damage to T.castaneum larvae,which was also consistent with the predicted results.To sum up,risk prediction and assessment experiments show that according to the factors and parameters that affect the efficiency of RNAi,we can predict the off-target effect of dsRNA on different genes,and it is applicable to different insects. |
PDF Full Text Request