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

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 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 editing designs. Either way, researchers can proceed directly to transformation after a one-hour isothermal reaction and recover the final construct within two days. By combining the advantages of sequence-ligation independent cloning (SLIC) cloning with a streamlined workflow, our approach facilitates rapid and efficient construction of CRISPR-Cas vectors and holds the promise of accelerating research and development in genome editing and related fields. To expedite the cloning of constructs, we propose a rapid one-step CRISPR-Cas vector assembly method that combines isothermal spacer removal with a sequence-ligation-independent cloning reaction. We could show that sothermal pacer emoval inearization and equence-igation ndependent loning (ISRL-SLIC) can create single, double and triple protospacer constructs in one reaction with scalability. The ISRL-SLIC reaction delivers clones under a broad range of oligo concentration making it a robust and time saving alternative to other methods for constructing CRISPR-Cas vectors.