产品介绍

Original Publication

EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I. Biotechnol J. 2016 Aug 11.; doi: 10.1002/biot.201600147. PubMed PMID: 27166612.

Description

The EasyClone-MarkerFree Vector Set is a complete kit for the introduction of genes into 11 predetermined EasyClone Chomosomal loci. These have been validated for stability and high expression. Each of these sites is located between essential genes and is thus not prone to being looped out of the chromosome. Each site can express up to two genes when combined with a bidirectional promoter (one on the sense and one on the antisense strand).

The kit comes complete with a Cas9 expression vector, 11 single site gRNAs, and 3 triple site gRNAs that guide the Cas9 to cut the chromosome in the specified chromosomal locations. These triple site gRNAs direct the Cas9 to cut in three separate loci, leading to the potential insertion of 6 genes per transformation (2 per locus). The double stranded break(s) are then repaired by the host cell using the native homologous recombination machinery, which incorporates the EasyClone-MarkerFree vector(s) into the target loci via double cross-over. This system is very efficient (90% correct clones with single insert, 60-70% with triple inserts) so there is no need for the insertion of markers into the host chromosome. This greatly speeds up the time to develop strains.

基本信息