TL;DR
A restriction enzyme-free and ligation-free PCR-based protocol widely applicable to any circular vector that enables targeted mutagenesis of the chikungunya virus (CHIKV) genome, offering a fast, efficient, and reliable strategy for generating mutant constructs suitable for virological and molecular studies.
A programmable gene replacement tool, named prime assembly (PA), which adapts prime editors to produce one or two pairs of 3'-flaps on both the genome and donor DNA, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest.
A streamlined cloning platform for expressing a gene of interest either alone or fused to a monomeric fluorescent protein under the control of two promoters of choice is described, providing a fast and flexible cloning strategy for protein expression and subcellular localization analyses in mycobacteria.
The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector, and includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing, and plasmid purification via miniprep.
These findings identify cloning-site proximity to an intact AAV ITR as a major determinant of plasmid stability during bacterial propagation and demonstrate that substantial loss of correctly assembled constructs can occur before AAV production.
This study demonstrates donor-complementary prime editing (DoPE) as a one-step, DSB-free and library-compatible method for precise insertion of large DNA fragments without requiring recombinases or transposases.
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