Rapid one-step CRISPR-cas vector assembly by isothermal spacer removal linearization and sequence-ligation independent cloning (ISRL-SLIC).

ABSTRACT: CRISPR-Cas genome editing is a powerful tool in various fields, but current cloning methods can be time-consuming due to the frequent use of intermediate entry vectors and multiple steps involving restriction enzymes and ligases. These multiple steps can create a bottleneck in CRISPR-Cas experiments. In response to this challenge, we propose a highly efficient streamlined approach, which enables simultaneous linearization of the acceptor plasmid and protospacer cloning in a single isothermal reaction. This eliminates the need for entry vectors, pre-linearization of vectors, and in vitro ligation, thus significantly simplifying the cloning process. The method can be applied to clone short synthetic oligos for single protospacer constructs or multiple amplicons for multiplex genome ed

SUBMITTER: Madsen CK 

PROVIDER: S-EPMC12395069 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature