genetic recording in yeast using crispr-cas9
TRANSCRIPT
Yeast Genome Editing Using CRISPR/Cas9
Robert Beem
Project Goal
Analyze activity of self targeting guide RNA (stgRNA) in Saccharomyces cerevisiae
Credit: Samuel Perli
Motivations
• Yeast grows faster amongst eukaryotes• Faster genomic evolution• Protein Evolution and Production• Study limitations of Cas9
Methods
• Plasmid Construction in E. coli (DH5α)
Antibiotic selection
Cas9 (S. pyogenes)
stgRNA
Methods
• Yeast Genome Integration
Homologous recombination
- Tryptophan, Leucine markers
W303-1A + Cas9 & stgRNA
Construction of Cas9 Plasmid
RBM1*
RBM3
RBM4
pTPGI dCas9 VP64
D10A H840A
H840AD10A
pTPGI dCas9TAA
pTPGI Cas9TAA
*starting plasmid
NLS
NLS
NLS
pTPGI Inducible Promoter
Ellis et al., Nature Biotechnology 2009
Gal + aTc TetR
pTPGICas9
Gal aTc Cas9Low Low Low
Low High Low
High Low Low
High High High
NLS
Construction of stgRNA PlasmidRBM2*
RBM7
RBM8
pRPR1 gRNA scaffold RPR t2micron originRBM2
pRPR1 gRNA scaffold RPR t
pRS405
pRPR1 stgRNA scaffold RPR tpRS405
SDS
TTGG AACC
*starting plasmid
Cas9
trp1
trp1-1
Cas9
trp1-1trp1
Homologous Recombination in Yeast
TRP: Bsu36ILEU: BfuAI
Cas9 Genome Integration
trp1-1: nonsense mutation, glu83STOP
RBY1
pTPGI Cas9TAA
W303-1A
Chr IV
stgRNA Genome Integration
leu2-3, 112: frameshift mutation, gly83
RBY2
W303-1A
Chr III
pRPR1 gRNA scaffold RPR tpRS405
SDS
TTGG AACC
Clontech, CRISPR_Cas9 2015
CRISPR/Cas Overview
3 steps:
1. Synthesis
2. Transcription
3. Targeting
CRISPR/Cas Synthesis
Natural:
Genome CRISPR/Cas gene
Artificial:
Integrated Plasmid stgRNA/Cas gene
CRISPR/Cas Transcription
Natural:
Bacterial machinery
Artificial:
Yeast machinery
CRISPR/Cas Targeting
Natural:
Cutting foreign DNA
Artificial:
SDS CCNNGG
PAM
stgRNA scaffold
SDS CCNNGG
PAM
SDS CCNNGG
PAM
NHEJ
Original DNA
Modified DNA
How do we assay Cas9 activity?
• Sequence mutations
• T7 endonuclease assay
New England Biolabs, Genome Editing 2015
T7 Endonuclease Assay
• Shows SDS editing has occurred
• Original Amplification: 1.4 kb
• Edited Amplifications:– 800bp– 600bp
Experimental Steps
Grow
• Gal +/- aTc• 22 hours
Amplify
• Forward primer 600bp upstream• Reverse primer 800bp downstream
Assay
• T7 endonuclease• Gel Electrophoresis
stgRNA T7 Endonuclease Assay(20bp SDS)
+ aTc- no aTc
Summary
• stgRNA is effective in S. cerevisiae
• pTPGI lacking aTc “leaks” Cas9
• Cas9 is significant in low quantities
Future Research
• Longer SDS• Expanding toolkit for genome engineering• Multiple promoters• Protein evolution using stgRNA• Applications:
– Protein engineering– Gene therapy