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