Journal: bioRxiv

Article Title: Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

doi: 10.1101/2021.12.29.474413

Figure Lengend Snippet: The feature of CRISPR/LbCas12a toolbox. a) the schematic diagram of pMM002P. The uracil phosphoribosyltransferase ( upt ) from M. maripaludis S2 as a counter-selective marker was co-expressed with the codon-optimized puromycin N-acetyltransferase ( pac ) under P mcr promoter (16). The promoter P hdr from Methanococcus voltae A3 was used to drive the expression of LbCas12a (25). The histone promoter P hmv from M. voltae A3 was used to express crRNA. Two PaqCI sties were placed between the direct repeat sequence and the synthetic terminator in the opposite direction for spacer fusion. The crRNA of the CRISPR tool plasmid pMM002P containing two PaqCI sites cannot target onto the native chromosome. An MreI restriction site was assigned between the promoters, P hdr and P hmv , for RF insertion if needed. b) cleavage test of pMM002P.

Article Snippet: All the cleavage plasmids were constructed as follow: (1) design the forward primer as “5’-AGAT+(23nt guide sequence)”, design the reverse primer as “5’-TATC+(23nt reverse complement guide sequence)” (2) anneal the two primers which contains the gRNA and have 4 nt overhang at 5’-end (3) ligate the annealing product with PaqCI (New England Biolabs) digested pMM002P.

Techniques: CRISPR, Marker, Expressing, Sequencing, Plasmid Preparation