Journal: bioRxiv

Article Title: A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

doi: 10.1101/2022.11.21.517425

Figure Lengend Snippet: a. Process for generation of long, linear ssDNA. Starting from a plasmid containing the insert of interest (IOI; blue), Golden Gate assembly is used to generate an intramolecular ligation product of the IOI. Undesired reaction products like backbone (gray) and concatemer plasmids of the insert are shown (Step 1). The two strands of the plasmid are labelled as T (top) and B (bottom), referencing the preference of the enzyme BbvCI. A cleanup step using BsaI-HFv2 and Exonuclease III removes undesired backbone reaction products (step 2). Then, circular ssDNA can be created from dsDNA using a nickase followed by exonuclease degradation (step 3). Finally, the cssDNA is linearized by Cas9 cleavage at the BbvCI recognition site, leaving a maximum of a 7 nt 5’ or 3’ scar (step 4). b. Plasmid design . The IOI (blue) is ligated into a backbone (grey) containing a high copy number ORI, a KanR resistance cassette, a BbvCI recognition site, and flanking BsaI-HFv2 recognition sites. The inset shows the sequence design of the adjacent BbvCI and BsaI-HFv2 recognition sites.

Article Snippet: In the pre-cycle step, 3 µL of 300 nM sgRNA diluted in NFH 2 O, 1 µL of 1 µM Cas9 Nuclease (diluted from 20 µM stock in 1X NEBuffer r3.1) (NEB; catalog # M0386T), and 1X NEBuffer 3.1 (NEB; catalog # B6003S) were combined in a 20 µL reaction volume, and the solution was placed in the thermocycler at 25°C for 10 minutes.

Techniques: Plasmid Preparation, Ligation, Sequencing

Journal: bioRxiv

Article Title: A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

doi: 10.1101/2022.11.21.517425

Figure Lengend Snippet: The linearization of the DNA using sgRNA- Spy Cas9 complexes was tested using 1 µg of M13mp19 dsDNA or ssDNA. a . Testing functionality of sgRNA. Spy Cas9 and designed sgRNA could cleave dsDNA only when both were included in the reaction (lanes 4-5). Proteinase K or 60°C incubation was used to dissociate the RNP from DNA. b . sgRNA- Spy Cas9 complex cleaves cssDNA with moderate efficiency. ssDNA cleavage using the sgRNA-Cas9 complex was successful but had poor yield. In order to properly visualize and differentiate linear ssDNA from circular ssDNA, the DNA was run on a 1.5% w/v gel with the addition of 60% v/v formamide followed by heating at 70°C for 5 minutes and then on ice for 5 minutes (to help denature the ssDNA and result in clearer bands). Variable temperatures did not increase yield.

Article Snippet: In the pre-cycle step, 3 µL of 300 nM sgRNA diluted in NFH 2 O, 1 µL of 1 µM Cas9 Nuclease (diluted from 20 µM stock in 1X NEBuffer r3.1) (NEB; catalog # M0386T), and 1X NEBuffer 3.1 (NEB; catalog # B6003S) were combined in a 20 µL reaction volume, and the solution was placed in the thermocycler at 25°C for 10 minutes.

Techniques: Incubation