Rapid Detection Of SARS-CoV-2 And D614G Mutation Based On CRISPR-Cas System

ObjectiveBased on the Clustered Regularly Interspaced Short Palindromic RepeatsCRISPR-associated(CRISPR-Cas)system,we established a simple,fast,highly sensitive,and highly specific detection method for SARS-CoV-2 and D614 G mutation.Methods1.The gene sequences of SARS-CoV-2 were compared and analyzed.We searched for the CRISPR-Cas12a detection targets in the conserved sequences of the open reading frame 1ab(ORF1ab)gene and nucleocapsid(N)gene of SARS-CoV-2.We also searched for the CRISPR-Cas13 a detection targets in the conserved sequences of N gene of SARS-CoV-2.2.The CRISPR-RNA(crRNA)and the DNA oligonucleotides with a T7 promoter sequence for crRNA were designed for different CRISPR-Cas detection targets.crRNA can be generated from DNA templates via in vitro transcription by T7 RNA Polymerase.3.The reaction systems of fluorescence detection based on CRISPR-Cas systems were established.The targets of ORF1 ab gene and N gene were screened by the CRISPR detection,and the target with the highest fluorescence value was selected as the final detection target.4.We designed primers for the targets of ORF1 ab gene and N gene to recombinaseaid amplification(RAA),and constructed the RAA reaction system.The best primers for targets were selected by RAA and DNA gel electrophoresis.5.The nucleic acid detection method for SARS-CoV-2 was established,which based on the RAA and CRISPR-Cas fluorescence detection.The specificity and the lowest detection limit were evaluated for the method.6.SARS-CoV-2 clinical samples and N gene simulation samples were used to verify the RAA-CRISPR based detection method for ORF1 ab gene and N gene of SARSCoV-2 that we established.7.According to above process that we described,we established the identification method for D614 G mutation which combined with RAA and CRISPR-Cas12a fluorescence detection.Results1.CRISPR-Cas12a based detection method for ORF1 ab gene of SARS-CoV-2 was established,which targeted ORF1ab-4.The primers for amplification of ORF1ab-4target are ORF1ab-4-AF5/ORF1ab-4-AR3.The CRISPR-Cas12a based detection method which target ORF1ab-4 had good specificity,and the lowest detection limit was one copy/μL within one hour.2.CRISPR-Cas12a and CRISPR-Cas13 a based detection methods for N gene of SARS-CoV-2 were established,that targeted N-4 and Cas13a-N-6.The primers for amplification of N-4 target and Cas13a-N-6 target are N-4-AF2/N-4-AR4 and Cas13aN-6-AF4/Cas13a-N-6-AR2 respectively.Both CRISPR-Cas12a and CRISPR-Cas13 a based detection methods which target N-4 and Cas13a-N-6 had good specificity,and the lowest detection limits were one copy/μL within one hour.3.CRISPR-Cas12a based detection method for D614 G mutation of SARS-CoV-2was established,that targeted the G614 strain.The primers for amplification of G614 target are G-AF5/G-AR1.The CRISPR-Cas12a based detection methods which target N-4 and Cas13a-N-6 had good specificity,and the lowest detection limits were 10copies/μL within one hour.Conclusions1.The simple,fast,highly sensitive,and highly specific detection methods for ORF1 ab gene and N gene of SARS-CoV-2 were established based on CRISPR-Cas12a and CRISPR-Cas13 a systems,which combined RAA with CRISPR fluorescence detection.2.A simple,fast,highly sensitive,and highly specific detection method for D614 G mutation of SARS-CoV-2 was established based on CRISPR-Cas12a system,which combined RAA with CRISPR fluorescence detection.