Research Of In Situ Hybridization For Breast Cancer HER2 MRNA Based On CRISPR/dCas9 Fluorescence Signal Amplification System In Fixed Cells And Tissues

RNA in situ hybridization can not only help us to observe the three-dimensional spatial structure of transcripts,explore the transcriptional mechanism and the distribution of varient RNA,but also can be used for clinical diagnosis of infectious diseases and cancers,even as the "gold standard".Depending on the detection method,ISH can be divided into chromogenic enzyme-based in situ hybridization and fluorescent in situ hybridization.Chemical reactions could always lead to diffusion and deposition of the substrate,which result in inaccurate localization of target nucleic acids.Although effective and accurate with high resolution,FISH requires specific fluorescent probes that are expensive,complex and time-consuming.Professor Doudna of the University of California,Berkeley found efficient CRISPR/dCas9-mediated gene editing In 2012.The application of CRISPR-based nucleic acid detection methods which showed great potential are also gradually emerging.The CRISPR-based genomic labeling relies on the formation of binary complexes of dCas9 and single guided RNA,target locus is recognized and detected by spacer sequence located on sgRNA.A PAM-presenting oligonucleotides(PAMmer)is introduced to replace PAM sequence and guide dCas9/sgRNA complexes in fluorescence labeling of specific RNA molecules.These two simple,fast and specific strategies make up for the shortcomings of mainstream RNA FISH methods.CRISPR/dCas9 based in situ hybridation can only be used for the labeling of high abundance RNA transcripts in living cells,which do not meet the requirement of clinical setting,because the signals are a little weak.Therefore,to make this tool more attractive and solve its intrinsic problem in clinical perspective,we incubate fixed cells and tissues with sgRNA from in vitro transcription reactions combined with purified dCas9,fluorescent labeled MS2 coat protein and PAMmer oligonucleotides to establish a novel single-molecule RNA in situ labeling method(RCasFISH),which exhibit highly sensitive and flexible.Current methods used to evaluate HER2 status of breast cancer include DNA FISH and immunohistochemistry.However,FISH require denatureation,the operation procedure is tedious and time-consuming and the probe is costly.In addition,the affinity of antibody is high for IHC,the result is subjective,and it needs to be interpreted by experienced physician.In this study,we first successfully purified dCas9 and MCP fusion proteins in vitro and designed PAMmers who are antisense to adjacent regions of the target mRNA and provide the protospacer adjacent motif(PAM)for Cas9 recognition.To increase signal intensity as well as signal-to-noise ratio(SNR),we extended sgRNA by inserting multiple tandem MS2 aptamers.We labeled the proteins with Halo ligands conjugated with Alexa Fluor 488(AF488)after purifying the recombinant fusion protein expressed in Escherichia coli.T7-synthesized sgRNA-MS2 stem-loop transcribed in vitro specifically allowed the enrichment of fluorescently labeled MCP at target HER2 RNA transcripts for the imaging.To evaluate the detection performance of the system,we evaluated the labelling efficiency,quantification ability,SNR,sensitivity,specificity,false positive ratio,reproducibility of RCasFISH and also compared it with commercially available smFISH as well as RNAscope kits.After that,mouse xenograft formalin-fixed paraffin embedded(FFPE)tissue samples and breast cancer FFPE tissue sections were used to evaluate clinical applicability by RCasFISH HER2 mRNA imaging.The results showed that RCasFISH could effectively label HER2 mRNA and low abundance RNA transcripts in fixed cells and tissues.Moreover,RCasFISH is sensitive to two or more mismatches between sgRNA and target mRNA but has tolerance for one mismatches.We got a good labeling efficiency(>85%)and signal-to-noise ratio as well as repeatability.In addition,RCasFISH can be used for single molecular quantification of intracellular transcripts including low abundance mRNA.Pairwise scatterplot showed a good linear relationship between RCasFISH and qPCR and correlation between them had an r=0.9259.Compared to traditional smFISH,our approach is brighter and has a higher signal-to-noise ratio.While compared with RNAscope,two-step RCasFISH can save time and effort.Of a total of 82 clinical breast cancer FFPE samples,including 43 HER2 positive,37 negative and 2 equivocal tissues,RCasFISH yield a concordance rate of 94.5%with FISH and IHC.The sensitivity of RCasFISH was 93.5%whereas the analytical specificity was 95.8%.Moreover,the positive predictive value was calculated to be 96.7%,which shows good test performance.Notably,RCasFISH could help distinguish the"HER2 equivocal" samples defined by FISH and immunohistochemistry,which has certain clinical molecular diagnostic value.In summary,we have developed and validated a rapid,sensitive,and efficient CRISPR-mediated single molecular RNA imaging tool for the detection of mRNA transcripts in fixed cells and FFPE tissue sections in the present study.Based on this RCasFISH,we have successfully achieved single-transcript resolution and completed the imaging and quantification of HER2 mRNA in clinical FFPE samples.It is worthnoting that RCasFISH has the potential to resolve FISH equivocal cases,providing an attractive alternative to traditional FISH or IHC.Additionally,this study also provides a new idea and technique for the imaging of oncogenic fusion gene transcripts and non-coding RNA using the multicolor imaging system.