Novel mad nuclease
A technology of nuclease and nucleic acid, applied in the field of nuclease
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Embodiment 1
[0067] Example 1: Overview of Exemplary Workflow
[0068] figure 1 An exemplary workflow 100 for creating and mining MAD series enzymes for in vitro screening is shown. In a first step 101, a vector is prepared and cloned to prepare a template vector into which the coding sequence of the mined MAD series RGN is inserted. In another step 103, metagenomic mining is performed to identify putative RGNs of interest based on eg sequence, potential PAM and likelihood of activity. After in silico identification of putative RGNs of interest, cassette 105 was constructed and cloned into a vector backbone, and then transformed into cells, resulting in a library of mined MAD series RGNs. Cells transformed with mined MAD series RGNs were arrayed in 96-well plates 107 for storage.
[0069] At step 109, an aliquot of cells from each well is taken, and the mined MAD series RGNs are amplified from each aliquot. In parallel, a gRNA library 110 was amplified for each mined MAD series RGN. ...
Embodiment 2
[0070] Example 2: Metagenome Mining
[0071] Metagenomes from different sources were used to assemble genomes (MAGs) including Genbank Bioproject accession numbers PRJNA348753, PRJNA385857, PRJNA432584 and PRJNA434545 to search for novel, putative CRISPR nucleases using HMMER Hidden Markov Models. Hundreds of potential nucleases were identified. figure 2 Novel RGNs discovered are shown, plotting protein size versus HMMER search score. For each MAG in the presence of a nuclease, putative CRISPR arrays were identified and spacer sequences were extracted. These spacers were then used as queries to search the JGI IMG / VR viral metagenomic database (Paez-Espino et al, Nucleic Acids Res. 2017 Jan 4;45(D1):D457-D465) and predicted based on viral sequence hits from adjacent spacers Putative PAM sequences. Thirteen nucleases (Table 1) were identified for in vitro validation based on sequence, potential PAM and confidence that the nuclease might be active. The sequence of each of ...
Embodiment 3
[0084] Example 3: Vector cloning, MAD series RGN library construction and PCR
[0085] The mined MAD series RGN coding sequence was cloned into the pUC57 vector with the T7 promoter sequence attached to the 5' end of the coding sequence and the T7 terminator sequence attached to the 3' end of the coding sequence. Transform 100 ng of the plasmid mix into E. SUPREME electrocompetent solo cells (Lucigen, Middleton, WI). After cells were recovered in 5 mL of recovery medium at 37°C for 1 hour in a shaking incubator, 1 mL of 50% glycerol was added, and cells were stored at -80°C in 100 μL aliquots.
[0086] Stored cells were diluted in phosphate buffered saline and spread on LB agar plates with 100 μg / mL carbenicillin. Cells were then grown overnight in an incubator at 37°C. Colonies were picked and inoculated into 1 mL of LB medium (100 μg / mL carbenicillin) in a 96-well culture block. Cultures were grown overnight at 37°C in a shaking incubator. Next, 1 μL of cells was dil...
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