Method and use for identifying plant species based on whole genome analysis and genome editing
A method for identifying plant species based on whole genome analysis and genome editing, i.e., a GAGE, includes that screening PAM-carrying sequences from a whole genome of a plant to be identified; aligning the PAM-carrying sequences with genomes of counterfeit species and closely related species, and then selecting sequences that are only present in the genome of the plant to be identified as target sequences; and performing a detection on the target sequences as selected by introducing a genome editing system, wherein based on the target sequences as selected, a CRISPR RNA (crRNA), which acts to guide a Cas protein to bind with a target sequence to form a complex, is designed and synthesized; then the trans-cleavage activity of the Cas protein is activated so that a single strand DNA bearing a fluorescent signal group is cleaved; and the plant species is identified by detecting the fluorescent signal.
1. A method for identifying plant species based on whole genome analysis and genome editing, the method comprising:
step 1, constructing a fragment genomic library based on a sequence of a whole genome of a first plant species,
wherein constructing the fragment genomic library in step 1 comprises: dividing the whole genome of the first plant species into L−K+1 fragments each having a length of K, the L−K+1 fragments constituting the fragment genomic library; calculating copy numbers of the L−K+1 fragments, and then determining genomic position of each fragment by aligning each fragment with the whole genome of the first plant species, wherein L represents a length of the whole genome and K represents the length of each fragment in the fragment genomic library;
step 2, constructing a candidate target sequence library by extracting candidate target sequences each carrying a protospacer adjacent motif (PAM) from the fragment genomic library;
step 3, aligning the candidate target sequences with whole genomes of at least one second plant species, and selecting any candidate target sequences that are only present in the first plant species as target sequences,
wherein step 3 further comprises: aligning the candidate target sequences obtained from step 2 with the whole genome of each at least one second plant species, wherein the target sequences as selected contain at least number (n) of different bases as compared with any sequence equal in length to the target sequences from the whole genome of each of the at least one second plant species, wherein n is greater than or equal to 3;
step 4, designing and synthesizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNAs based on the target sequences;
step 5, extracting genomic DNA of a plant to be detected wherein the plant to be detected is a plant sample of the first plant species, the second plant species, or a species other than the first plant species and the second plant species;
step 6, amplifying and recovering the target sequences as a DNA substrate or directly using the extracted genomic DNA of the plant to be detected as a DNA substrate;
step 7, selecting a genome editing system selected from the group consisting of a CRISPR/Cas12a system or a CRISPR/Cas13a system;
step 8, according to a selected genome editing system of step 7, carrying out a reaction with at least six (6) ingredients comprising: a buffer, a CRISPR-associated protein (Cas), the CRISPR RNAs (crRNAs), nuclease-free water, the DNA substrate of the plant to be detected, and single-stranded DNA (ssDNA) fluorescent reporter gene;
step 9, performing a fluorescence detection, wherein a Cas is guided by the crRNAs to recognize and combine with the target sequences to form a ternary complex, the trans-cleavage activity of the Cas is activated to cut the ssDNA fluorescent reporter gene, such that an emitted fluorescence can be detected; and
step 10, determining that the species of plant to be detected is the first plant species when a result of the fluorescence detection has a significant difference of P<0.01 from a blank control without the DNA substrate, and otherwise, determining that the species of the plant to be detected is not the first plant species.
2. The method according to claim 1 , wherein step 2 further comprises screening for PAM motif in each fragment of the fragment genomic library and constructing the candidate target sequences library by extracting the candidate target sequences carrying the PAM.
3. The method according to claim 1 , wherein n is adjusted.
4. The method according to claim 1 , wherein step 5 further comprises: amplifying the genomic DNA of the plant to be detected with primers which specifically amplify the target sequences, and recovering the target sequences as a DNA substrate; or, amplifying the genomic DNA of the plant to be detected with primers which specifically amplify DNA sequences containing the target sequences, and recovering the DNA sequences containing the target sequences as a DNA substrate.