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Exploiting CRISPR-Cas nucleases to produce sequence-specific

antimicrobials

David Bikard

Synthetic Biology Group

13/12/2016

The CRISPR adaptive immune system

Clustered Regularly Interspaced Short Palindromic Repeats

S. pyogenes CRISPR02

tracrRNA

Deltcheva & al., Nature 2011Jinek & al., Science 2012

CRISPR as a biotechnological tool

Homologous Recombination

NHEJ

Small indels

Point mutationGene insertion

etc.

No repair

Cell death

Cas9

NHEJ = Non-Homologous End Joining

• S. pneumoniae (Jiang, Bikard et al, 2013, Nat. Biotechnol.)

• E. coli (Jiang, Bikard et al, 2013, Nature Biotechnol.)

• Human cells (Le Cong et al., 2013, Science)

• Monkeys (Niu et al., 2014, Cell)

• Livestocks (Tan et al., 2013, PNAS)

• Mice (Wang et al., 2013, Cell)

• Frogs (Blitz et al. and Nakayama et al., 2013, Genetics)

• Zebrafish (Hwang et al., 2013, Nature Biotechnol.)

• Insects (Wang et al., 2013, Cell. Res.)

• Plants (Li et al. , Shan et al., 2013, Nature Biotechnol.):

• Flies (Gratz et al., 2013, Genetics)

• Nematodes (Lo et al., 2013, Genetics)

• Yeast (DiCarlo et al., 2013, NAR)

Genome Editing

Homologous Recombination

NHEJ

Small indels

Point mutationGene insertion

etc.

No repair

Cell death

Cas9

CRISPR as a biotechnological tool

Sequence-specific killing of S. aureus

pDB114

Bikard & al., Nat Biotech, 2014

Plasmid transduction

Sequence specific killing


0

5000

10000

15000

20000

25000

30000

35000

40000

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 200 400 600 800 1000

KanR

KanS

Competition with non-targeted strain

OD

(6

00

nm

)

GFP

Time (min)


Sequence specific killing of MRSA (USA300) in a mixed population

Spacer

Cm

Selection

OxaØ

mecA

Ø

Ø

mecA

Ø

mecA

10-2 10-3 10-4

Dilution

10-1100

Plasmid curing in USA300

100

101

102

103

104

105

Ø 1 2 1+2

Ø

Cm

Tet

pUSATarget:

Selection

CF

U/u

l

pUSA01

1 2 3 4 5 6 7 8

pUSA02

C

- 0.5kb

- 1kb

- 0.5kb

- 1kb

Strain immunization

Mice experiments

Chad Euler, RockefellerVince Fischetti, Rockefeller

5’ 3’

5’3’

RuvC

HNH

20nt

*

*

Catalytically dead Cas9 (dCas9)

dCas9-mediated repression

Qi et al, Cell. 2013

Bikard* , Jiang* & al., NAR, 2013

Inhibition of

transcription

initiation

Inhibition of

transcription

elongation

Qi et al, Cell. 2013


dCas9-mediated repression

dCas9-mediated activation



23 fold

7 fold



• Library construction through on-chip oligo synthesis and cloning

• Perform functional screen

Sub-inhibitory antibiotic Abiotic stresses

Query gene knockout / overexpression…

• Infer the fitness of each knockout by measuring changes in the proportion of guide RNAs in the library

CRISPR screens

Before the experiment Before the experiment

Aft

er

the

exp

eri

me

nt

Aft

er

the

exp

eri

men

t

No induction + aTc

High throughput dCas9 screens

Before the experiment Before the experiment

Aft

er

the

exp

eri

me

nt

Aft

er

the

exp

eri

men

t

No induction + aTc

Depletion of gRNA targeting essential genes

murA

Target on the template strand Target on the coding strand

The Synthetic Biology Group

Team• Florence Depardieu • Lun Cui • Gayetri Ramachandran • Aude Bernheim• Belen Gutierrez-Soriano• Antoine Vigouroux• Alicia Calvo Villamanan

Collaborations• Luciano Marraffini, Rockefeller• T. Lu, MIT• E. Rocha, Institut Pasteur• D. Mazel, Institut Pasteur• S. van Teeffelen, Institut Pasteur• Bruno Dupuy, Institut Pasteur• Paul Planet, Columbia University• Pamela Schnupff, Institut Imagine

Funding

LabEx IBEID

How many phagemids to kill a cell?

What happens to survivors ?

- Some cells are less likely to receive phagemids than others ?

- The phagemid is lost through segregation before it kills the cells ?

- Cells receive a defective CRISPR system ?

- Mutation at the target sequence


Escapers

Out of 8 surviving colonies:- 6 are sensitive to ChloramphenicolThey either lost the phagemid or did not receive it- 2 received a defective Cas9

All are still sensitive to the treatment The target is still intact

Future directions

• How are DNA breaks introduced by CRISPR processed in bacteria ?

Lun Cui

Future directions

• How are DNA breaks introduced by CRISPR processed in bacteria ?

Lun Cui

SOS response Large deletions

• Can DNA repair interfere with CRISPR immunity?

Future directions

Aude Bernheim

Palmer, K. L., & Gilmore, M. S. (2010). mBio, 1(4)

Enterococci with CRISPR accumulate less antibiotic resistances

What could explain the apparent lack of trade-off?

• Mobility of CRISPR-Cas systems

• CRISPR might actually most beneficial in organisms undergoing high HGT rates

• Transcription dependent interference

• Spacer acquisition is not random (priming)

Telltale marks of CRISPR

inactivation in available genomes

Characterization of the PAM sequence

Jiang*, Bikard* & al., Nat. Biotech., 2013

CRISPR+

CRISPR-

Transformation

Bad PAM motifs

Everything

Target NNNNN

Library of randomized PAM sequences

PAM requirements

A AAA 1.02 ACA 1.06 AGA 0.95 ATA 1.02

C CAA 1.03 CCA 1.03 CGA 0.97 CTA 1.00

G GAA 1.10 GCA 1.09 GGA 0.93 GTA 1.01

T TAA 1.02 TCA 1.11 TGA 0.91 TTA 1.11

A AAC 1.07 ACC 1.02 AGC 1.00 ATC 1.09

C CAC 1.06 CCC 1.04 CGC 1.00 CTC 1.09

G GAC 1.05 GCC 1.02 GGC 1.02 GTC 1.22

T TAC 1.06 TCC 1.05 TGC 1.03 TTC 1.06

A AAG 0.49 ACG 0.97 AGG 0.06 ATG 0.96

C CAG 0.78 CCG 1.08 CGG 0.06 CTG 1.03

G GAG 0.50 GCG 1.00 GGG 0.06 GTG 1.20

T TAG 0.86 TCG 0.91 TGG 0.07 TTG 1.06

A AAT 1.01 ACT 1.09 AGT 1.04 ATT 1.03

C CAT 1.03 CCT 1.03 CGT 1.07 CTT 1.08

G GAT 1.04 GCT 1.08 GGT 1.08 GTT 1.12

T TAT 1.06 TCT 1.03 TGT 1.06 TTT 1.01

1st P

AM

po

sitio

n 3rd P

AM

po

sitio

n

A

C

G

T

2nd PAM position

A C G T

R=N° of reads after transformation in CRISPR+ strain

N° of reads after transformation in control strain


Target requirements


Adaptation against phage challenge

PAM motif

Heler & al., Nature 2015

Cas9 specifies functional viral targets

Swapped Cas9

Heler & al., Nature 2015

exploiting crispr-cas nucleases to produce sequence ... · • florence depardieu • lun cui •...

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