CRISPR Constructs and Deletions of BUL1, BUL2, Jen1, Art4 in
Saccharomyces cerevisiae
Dr. Evan Guiney, PhD, Elaina Natali, Morgan Morrison

Background
Art4, an alpha arrestin and adaptor protein, is involved in
ubiquitin-dependent endocytosis of the Jen1 pump. In
addition, Bul1 and Bul2 exhibit activity in the endocytosis
of Jen1
Art1-Mup1 is the one of the best adaptor-pump pairs
understood.
Art4-Jen1 is the best candidate for characterizing a second
adaptor-pump pair.
Delete genes that encode these proteins to conduct a
genetic screen.

Art4 PCR did not yield desired products needed for
Art4 deletion
Bul1/Bul2 repair fragment successfully created using
PCR
CAS9 Expression Fragment
CRISPR constructs along with the repair fragments
were put into yeast cells from 2 different strains and
growth was observed.

Phusion
Polymerase

Ladder
Art4 Repair (left-long)
Art4 Repair (left-short)
Art4 Repair (right-long)
Art4 Repair (right-short)
Phusion polymerase
Phusion polymerase

Findings

Ladder
BUL1 Cut Fragment
BUL1 Repair Fragment
Cas9 Expression Fragment

Taq Polymerase

Death

Pro

Coding Region (ART4 or BUL1)

Ter

Deletion of Coding Region

Future Studies
Troubleshoot for Art4 deletion by varying magnesium
concentration, annealing temperature. Try new
genomic DNA preparation or ordering new primers if
needed.
PCR characterization of colony growth amongst the
two strains to check for correct integration location of
the CRISPR repair
https://benchling.com/pub/ellis-crispr-tools