Establishment And Application Of CRISPR/Cas Based Detection For Cryptosporidium And Enterocytozoon Bieneusi

Cryptosporidium spp.is a kind of parasitic protozoan that can infect the gastrointestinal tract of many animals and human,with C.parvum and C.parvum ?d subtypes being the most popular species and subtypes,respectively.Enterocytozoon bieneusi is the Microsporidia species that has been mostly reported.In addition to infecting humans,E.bieneusi can infect wild animals,domesticated livestock and poultry,companion animals,and so on.Being infected with these intestinal protozoa,individuals with normal immune function usually present with asymptomatic infection or self-limiting diarrhea,while infections in immunocompromised individuals can lead to severe disease and even death.These pathogens pose a great threat to public and human health.Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a repeating sequence in the genome of a prokaryote that can transcribe CRISPR RNA(crRNA)with specific sequence and produce CRISPR-associated(Cas)proteins.The Cas proteins can identify,combine to and cleave the target DNA/RNA leaded by homologous crRNA.According to the difference among Cas proteins,the CRISPR/Cas system have been harnessed to gene editing and nucleic acid detection.In the present study,recombinant protein LwCasl3a was successfully solubly expressed in RosettaTM(DE3)induced by IPTG.Through a series of optimization tests,the prokaryotic expression conditions were,determined as follows:the final concentration of IPTG was 0.5 mM,and induced for protein expression at 18? for 16 hr.In this study,the recombinant protein LwCasl3A was purified by Ni column affinity chromatography using the 6 × His fusion label.The optimal purification conditions of recombinant protein LwCasl3a were determined by imidazole elution with gradient concentration as follows:miscellaneous proteins were washed away by 40 mM imidazole and the recombinant protein LwCasl3a was eluted by 500 mM imidazole.The recombinant protein LwCas13a with high puity was finally purified using the AKTA purifier.The high bioactivity of LwCasl3a was reserved by soluble expression and the unique characters and applications of Cas13a can be explored using LwCasl3a purified here,which also lay the foundation for the follow-up researches.In this study,a specific nucleic acid fragment of the SSU rRNA gene of C.parvum was selected as the target sequence,according which the homologous crRNA of LwCas 13a was designed.Two reverse complementary single strand crDNA were synthesized and annealed as a double strand crDNA,which was then transcribed as crRNA using T7 in vitro transcription.By integrating Recombinase Polymerase Amplification(RPA),T7 in vitro transcription and CRISPR/Cas13a trans-cleavage,we established a "OnePot" rapid detection method of C.parvum,having a convenient way to present the results which can be read by fluorescence intensity recorded by universal fluorescent equipment or by fluorescence signal under blue light viewed by naked eyes.The specificity test showed that this method had good specificity and did not cross-react with other species of Cryptosporidium or other common intestinal parasitic protozoa.The sensitivity test showed that the lowest oocyst concentration that could be detected by this method from feces was 10 oocysts per gram,indicating a high sensitivity.This detetion method also corroborated 100%with the conventional PCR-sequencing method by applying in the same batch of clinical samples.The"One-Pot"diagnosis of C.parvum established in this study is a rapid detection method with high specificity,sensitivity and robustness,which can be applied in the on-site veterinary clinic,moreover,it also has broad application prospects in resource-deficient areas.In this study,by align the sequences of the gp60 gene with different subtypes of C.parvum,we screened out a ?d subtypes-specific fragment as the target sequence,and designed the homologous crRNA of FnCas12a.The crRNA was prepared by T7 in vitro transcription and purified to be with high purity.By integrating recombinase polymerase amplification and Casl2a/crRNA trans-cleavage(termed RECTC),we established an end-point diagnostic by observing fluorescence readouts by naked eyes under blue light and an on-site diagnostic using lateral flow strip.Our ReCTC based diagnoses could detect the ?d subtype family of C.parvum from clinical fecal samples independent of professional technicians,expensive instrument or cumbersome operation.The newly established RECTC based detection can detect 1.9×1018 M recombinant plasmid containing gp60 fragment of ?d subtypes or 10 oocysts per gram feces.The specificity has been also confirmed to be robust and has no cross-reaction with other subtypes of C.parvum,other species of Cryptosporidium or other common intestinal protozoa.The RECTC based detection also corroborated 100%with the conventional PCR-sequencing method,and can be used in the diagnosis of C.parvum ?d subtypes in clinical samples.In this study,by integrating recombinase polymerase amplification and Casl2a/crRNA trans-cleavage(termed RECTC),we established a rapid diagnosis of specific nucleic acid fragment of E.bieneusi.The results can be indicated by the fluorescence signal observed by the naked eyes under the blue light or by the test line on the lateral flow strip.The RECTC based detection of E.bieneusi was an on-site diagnostic independent of professional technicians,expensive instrument or cumbersome operation.This diagnostic has a limit of detection(LOD)of 3.7 copies/reaction of E.bieneusi DNA,and has no cross-reaction with other common intestinal protozoa,indicating a high specificity and sensitivity.The application of the RECTC based detection in clinical samples confirmed the higher reliability than conventional PCRsequencing methord based on the Internal Transcription Spacer(ITS)sequence,and can be stably used in the detection of E.bieneusi in clinical samples.In this study,the newly established rapid detection methods of intestinal protozoa based on CRISPR/Cas technology was successfully applied to the detection of large quantities of clinical samples,and was compared with the PCR-sequencing method and superior to the latter.The positive rate of Cryptosporidium was relatively high on the dairy cattle farm selected in Zhongmu,Zhengzhou.And E.bieneusi infection was also observed,suggesting the need to strengthen the feeding management as well as the prevention and control of these parasites.