P045
Prokaryotic immune system in eukaryotes: type I-E
CRISPR-system in Saccharomyces cerevisiae
Lina Amlinger, Devashish Rath and Magnus Lundgren
Department of cell and molecular biology, Uppsala,
Sweden
The type I-E CRISPR system is one of the most studied CRISPR
systems so far and has been extensively studied in different E.
coli strains. For immunity, Cas3, crRNA and the large protein
complex Cascade are required. Cascade, composed of five types
of proteins, processes the pre-crRNA and, when loaded with
crRNA, acts as a guide to target the invading DNA for destruction.
Cas1 and Cas2 have been shown to be required for spacer incorporation, but are not essential for immunity.
Type II systems have been used for genome editing in
mammalian cells. This requires expression of the large Cas9protein and a fusion crRNA-tracrRNA. However, interference
against mobile genetic elements has not been demonstrated in
eukaryotes, nor has spacer incorporation.
We are currently working towards reconstituting all stages of type
I-E CRISPR immunity in Saccharomyces cerevisiae; adaptation,
expression and interference. The Cas-genes and the CRISPR
array have been cloned under separate inducible promoters
in yeast vectors. The interference of the system is tested by,
primarily, transformation efficiency experiments while expansion
of the CRISPR array is tested through PCR. This would be the
first time all stages of CRISPR immunity is reconstituted in a
eukaryotic cell.