Gene regulation

via cas9 proteins

NMCAS9 & ST1CAS9

CRISPR-associated systems
Bacterial DNA with integrated viral DNA fragments

Transcription of DNA fragment

cas9

Incoming
foreign plasmid

CRISPR-associated systems
Bacterial DNA with integrated viral DNA fragments

Transcription of DNA fragment

cas9

Incoming
foreign plasmid

Repurposing the CAS system

ST1dcas9

ST1cas9 from
streptococcus thermophilus
PAM: NNAGAAW
W denotes either A or T.
sgRNA handle is ~ 125 b.p.

NMdcas9

SPdcas9

NMcas9 from neisseria

meningitides
PAM: NNNNGATT
sgRNA handle is ~101 b.p.

SPcas9 from
Streptococcus pyogenes
PAM: NGG
sgRNA handle is ~ 120 b.p.

sgRNA Handles

ST1cas9

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Esvelt et. al

ST1cas9

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Esvelt et. al

Repurposing NMcas9 & ST1cas9 as

Transcription Factors
1)
ST1cas9

2)
ST1dcas9

3)
ST1dcas9

Induce mutation in
nuclease catalytic region

Engineer sgRNA to attain

directed protein-DNA binding

Attach Nuclear Localization

Sequence to dcas9 protein

ST1dcas9

ST1dcas9

NLS
ST1dcas9

Repurposing NMcas9 & ST1cas9 as Transcription Factors

Can attach
1)Activation domains, ex. VP64

3) Degrons, ex.truncation 1(T1) of IAA17

Auxin induced

T1

AD

ST1dcas9

AD

Promoter x Target1

ST1dcas9
Promoter x Target1

2)Repression domains, ex. TUP1 or Krab

RD

ST1dcas9
Promoter y Target2

Lu system
AD

ST1dcas9
AD

ST1dcas9

Finite State Machines

FSM Parts
AD

ST1dcas9

T1

AD

NMdcas9

AD

SPdcas9

SPdcas9

RD

Next Steps
Test Lu System using ST1cas9 and NMcas9
Experiment with repression domains optimize
repression
Continue to optimize degron tags with the
dcas9 proteins
Integrate completed tool kit into a finite state
machine

Sources

Orthogonal Cas9 proteins for RNA-guided gene regulation and

editing (Church, 2013)

Efcient genome engineering in human pluripotent stem cells using

Cas9 from Neisseria meningitides (Thomson, 2013)

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription

in Eukaryotes (Qi, 2013)

Repurposing CRISPR as an RNA-Guided Platform for SequenceSpecific Control of Gene Expression (Lim, 2013)

Framework for Engineering Finite State Machines in Gene Regulatory

Networks (Kevin Oishi and Eric Klavins 2013)

Parts Slide
AD

RD

ST1dcas9

Promoter 1

NMdcas9

gRNA1

SPdcas9

gRNA2
Promoter 2

Promoter 3

Target1
Promoter x

AD

gRNA3

Target2

Promoter y

Target3
Promoter z

Next Steps

Express each dcas9s 7 sgRNAs off of 2micron plasmids increase

expression

Add MXi1 and Gal80 to dcas9 proteins