Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.

ABSTRACT: There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is prov

SUBMITTER: Rajkumar AS 

PROVIDER: S-EPMC8800938 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature