Expanded CRISPEY Kit (Kit #1000000257)

 Depositing Lab : Jeffrey Lewis

Expanded CRISPEY Kit은 Saccharomyces cerevisiae(효모)에서 유전체 편집을 수행하기 위한 플라스미드 세트로, CRISPR/Cas9 절단과 세포 내 발현되는 template에 의한 homologous recombination을 결합하여 작동합니다.

이 시스템을 이용하면 deletion, point mutation, insertion 등 다양한 변이를 만들 수 있습니다.

이 키트는 실험실 균주, 야생 균주, 산업 균주 모두에서 사용할 수 있으며, haploid와 diploid모두 적용 가능합니다. editing 효율은 50%에서 95% 이상에 이릅니다.

본 키트는 96-well plate format의 bacterial glycerol stocks으로 제공됩니다.

Description 

 The Expanded CRISPEY Kit contains six pCRISPEY plasmids, seven pCas9-EcRT plasmids, and five control plasmids. Each pCRISPEY plasmid contains a URA3 gene for auxotrophic selection and one of three antibiotic selection markers (Kanamycin, Hygromycin, or Nourseothricin). Each pCas9-EcRT plasmid contains one of four selectable markers (Kanamycin, Hygromycin, Nourseothricin, or HIS3). Two versions of each pCRISPEY and pCas9 plasmid are available with different induction systems (galactose-inducible or estradiol-inducible). The pCRISPEY control plasmids target the ADE2 gene.

The pCRISPEY plasmids contain a unique cloning site: NotI for galactose-inducible plasmids or XhoI for estradiol-inducible plasmids. A user-designed guide RNA (gRNA) and repair template (obtained separately) can be synthesized as a single oligo and cloned into the pCRISPEY vector. This cloning strategy typically has >95% cloning efficiency. The pCRISPEY plasmid is then co-transformed into yeast with a pCas9-EcRT plasmid. Upon induction, the gRNA and repair template are expressed from the pCRISPEY vector while the Ec86 reverse transcriptase and SpCas9 are expressed from pCas9-EcRT. The Ec86 reverse transcriptase amplifies the gRNA and repair template in vivo to increase the available template for homologous recombination, yielding high editing efficiencies (50–99%). Plasmids can be 'cured' from final strains to generate markerless genome edits, which would also enable making multiple edits sequentially.

Figure 1: The CRISPEY system pairs a pCRISPEY plasmid with a pCas9-EcRT plasmid for directed editing of the yeast genome. User-designed gRNA and homologous repair template are cloned into pCRISPEY at a unique NotI (for galactose-induced plasmids) or XhoI (for estradiol-induced plasmids) site.

Kit Documentation

CRISPEY-kit-manual (PDF, 2.3 MB)

Contents of Kit