Novel CRISPR/Cas12f enzymes and systems

[0216]The acquisition of embodiment 1.Cas12f gene and Cas12f guide RNA

[0217] 1. CRISPR and gene annotation: use Prodigal to annotate the microbial genome and metagenomic data of NCBI and JGI databases to obtain all proteins, and use Piler-CR to annotate CRISPR loci, and the parameters are default parameters.

[0218] 2. Protein filtering: De-redundant annotated proteins through sequence consistency, remove proteins with completely identical sequences, and classify proteins longer than 800 amino acids into macromolecular proteins. Since all the effector proteins of the second type of CRISPR / Cas system discovered so far are more than 900 amino acids in length, in order to reduce the computational complexity, we only consider macromolecular proteins when mining CRISPR effector proteins.

[0219] 3. Acquisition of CRISPR-related macromolecular proteins: Extend each CRISPR locus upstream and downstream by 10Kb, and identify non-redundant macromolecular proteins in the adjacent C...

[0224] Example 2.Cas12f gene processing of mature crRNA

[0225] 1. Artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO:4, and simultaneously artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO:10.

[0226] 2. Ligate the double-stranded DNA molecule synthesized in step 1 with the prokaryotic expression vector pACYC-Duet-1 to obtain the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f.

[0227] The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f was sequenced. Sequencing results show that the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f contains the sequences shown in SEQ ID NO:4 and SEQ ID NO:10, and expresses the Cas12f.4 protein and SEQ ID NO:1 shown in SEQ ID NO:1 Cas12f.4 prototype direct repeat sequence shown in NO:7. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f was introduced into Escherichia coli EC100 to obtain a recombinant bacterium, which was named EC100-CRISPR / Cas12f.

[0228] 3. Take a single clone of EC100-C...

[0239] PAM domain identification of embodiment 3.Cas12f gene

[0240] 1. Construct the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f and sequence it. According to the sequencing results, the structure of the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f is described as follows: replace the small fragment between the recognition sequences of the restriction endonuclease Pml I and Kpn I of the vector pACYC-Duet-1 with SEQ ID NO : A double-stranded DNA molecule shown at positions 1 to 3713 from the 5' end in the sequence shown in 4. The recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f expresses the Cas12f.4 protein shown in SEQ ID NO:1 and the Cas12f guide RNA shown in SEQ ID NO:25.

[0241] 2. An expression cassette is contained in the recombinant plasmid pACYC-Duet-1+CRISPR / Cas12f, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:23. In the sequence shown in SEQ ID NO:23, the 1st to 44th from the 5' end is the nucleotide sequence of the pLacZ promo...