Work

A method for generating user‐defined circular single‐stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

Year: 2023

Type: article

Abstract: Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.

Source: Biotechnology and Bioengineering

DOI: 10.1002/bit.28471

Authors: Isabell K. Strawn, Paul J. Steiner, Matilda S. Newton, Zachary T. Baumer, Timothy A. Whitehead

Institution: University of Colorado Boulder

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Institutes of Health, National Institute of General Medical Sciences

Award: Engineering Long ssDNA for Genome Editing Applications