defining and characterizing the components of a crispr-cas9 … · crispr medicine guide + sequence...

1© 2017 Editas Medicine© 2017 Editas Medicineeditasmedicine.com

Defining and characterizing the components of a CRISPR-Cas9

genomic medicineHari Jayaram

CRISPR International Conference 2017June 10, 2017

2© 2017 Editas Medicine

Presentation Overview

Sickle Cell Disease, b-globin Gene Regulation, and Editing Strategy

RNP based Lead Discovery platform

Fully synthetic modular dgRNA

Ex-vivo T-cell and HSC editing with Cpf1

Platform for CRISPR medicines


Pipeline strategy to enable successful medicines


Scalable, Consistent Engineered Cell Therapies

Engineer multiple components to T-cell & HSC sensitivity (maintain cell viability, potency)

Platform for CRISPR Medicines

EngineeredPatient Cell

PatientCell

RNA GuidedEndonuclease

RNAGuide

Ribonucleoprotein Particle (RNP)

+

Electroporation


Aspects of a Ribonucleoprotein medicine

Defining every component : key to a successful CRISPR medicine

Guide Sequence

+ =

• Efficacy: well optimized cellular and well characterized in vitro efficacy

• Stability: track RNP activity through gene-editing workflow and therapeutic window

• Fidelity: Define every component and characterize trace elements

• Pharmacokinetics





Fully synthetic modular gRNA




RNP lead discovery

▪ Wealth of standardized data across cell types, nucleases, gRNA formats

T1 T3 T4 T5T2

Many factors influence gRNA efficacy: gRNA sequence, Nuclease type, cell type

8© 2017 Editas MedicineCONFIDENTIAL

T-cells skew to deletions as compared to insertions in HEK-293T cellsCell type dependence

BCL11Ae−CUNAM−FUGY BCL11Ae−FUVOJ−LEDA BCL11Ae−GEKOD−NYDO BCL11Ae−GUDUN−XORA

BCL11Ae−HUKAT−PAPA BCL11Ae−HUSYB−SALA BCL11Ae−HYFIH−VOMI BCL11Ae−KAQYV−GEZY

BCL11Ae−LUQUG−CISY BCL11Ae−MUXID−TYGE BCL11Ae−NAQOW−VUNA BCL11Ae−PIDAR−GIXU

BCL11Ae−QABOV−KOZA BCL11Ae−QUTYJ−GIRA BCL11Ae−RETEQ−CEFU BCL11Ae−VAJOT−JEGI

BCL11Ae−XOVIS−GUHA BCL11Ae−ZUJUP−LEBA

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−60 −40 −20 0 20 40 −150 −100 −50 0 50 −100 −50 0 50 −25 0 25 50

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−50 −25 0 25 50 −50 −25 0 25 50 −25 0 25 50 −25 0 25 50

−200 −150 −100 −50 0 50 −30 0 30 60 −25 0 25 50 −200 −150 −100 −50 0 50

−150 −100 −50 0 50 −250 −200 −150 −100 −50 0 50Size

dens

ity

Cell.TypeHEK293T cell

Edit change in size (bp)

InsertionsDeletions

Prob

abilit

y de

nsity

Cell type

Guide 1 Guide 2 Guide 3

Guide 4 Guide 5 Guide 6


Normalized indel distributions display range of levels of control

Guide dependence of edit

More control Less control

Normalized edit profiles

10© 2017 Editas Medicine© CONFIDENTIAL 2016 Editas Medicine

▪ Spy-Cas9 shows the +1 insertion more often than Spy-Cas9

Indel pattern dependence on nuclease choice

10

%age of indelswith a specific edit

+1 insertions


Pyogenes cuts with a 1bp stagger more often than Sau Cas9

+1 overhangs a result of nuclease properties

In vivo genome editing using Staphylococcus aureus Cas9.Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F.Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299.

+1 insertions come from base 5’ of cut site

https://www.ncbi.nlm.nih.gov/pubmed/25830891


Maturation of the synthetic gRNA field allow for multiple formats

▪ Chemical synthesis of gRNA allows for greater flexibility, fidelity , scale and purity

▪ Synthetic chemistry goes from 3’ to 5’ , 100-mers were challenging till recently

▪ Safety and Specificity of a medicine rely on a precise characterization and control of every component

gRNA component at the core of the RNP medicine

Guide Sequence

100-mer sgRNA


5’ 3’

5’

3’

5’

3’

+

Why make a synthetic guide?

• Targeted chemistries anywhere in the molecule

• Unhindered ends and modifications

• Scale up and purity are more compatible with CMC requirements

Generating Synthetic Covalently-Coupled Dual gRNA

A completely non-enzymatic process for guide production

covalently-coupled dual gRNA (dgRNA)


Cellular Editing ActivityIn vitro transcribed and synthetic covalently-coupled dgRNA are equivalent in cells

-11 -10 -9 -8 -7 -60

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Log concentation RNP (M)

% E

ditin

g (T

7E1)

IVT purified by vendorIVT purified by collaborator

unligated 2 part syntheticligated 2 part synthetic

IVT purified by vendor

covalently-coupled dgRNAIVT purified by collaborator

2-part synthetic


gRNA purity and sequence fidelity

A

B

Covalently-Coupled dgRNA

Covalently-coupled dgRNA result in greater sequence fidelity in target region

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−3 −2 −1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Position

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geBase

ACGT+−

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enta

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BaseACGT+−


gRNA Purity and Sequence Fidelity

A

B

Covalently-Coupled dgRNA

Covalently-coupled dgRNA result in fewer truncated molecules


▪ Smaller gRNA just 44 nucleotides▪ The business end of gRNA is at 3’▪ dsDNA Cutting profile different from Spy and Sau Cas9▪ More targets with enzyme and evolved variants

Type VI nuclease Cpf1 with many benefits

Cpf1

- WT TTTV, TTN PAMs plusTYCV/CCCC, TATV engineeredPAM variants

- Single guide with 20-24 ntprotospacer and 19-20 nt direct repeat (~40 nt)

- 5’ staggered DNA cut with 4-5 ntoverhangs

Cas9

- WT NGG PAM plus NGAN, NGCG engineered PAM variants

- Separate crRNA and tracrRNA that can be linked into one molecule (~100 nt)

- Blunt DNA cut


AsCpf1 emerging as the “go to” Cpf1 with Robust activity

Screening of multiple Cpf-1 orthologs and variants

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MS1 MS5 MS11 MS18

% in

dels

by

T7E

1% indels at four matched sites in U2OS

AsCpf1 FnCpf1 LbCpf1 Lb2Cpf1 SpCas9


Cpf1 editing at T-cell targets

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% in

dels

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T7E

1

% indels at a targeted locus


Sample editing by AsCPf1 in HSCs

Ex-vivo editing of Cpf1 at sample locus

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MS1 MS5 MS11 MS18

% in

dels

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S

% indels at four matched sites

AsCpf1 SpCas9


Summary and Conclusions

▪ A flexible platform allows for lead discovery screening and optimization at scale

▪ Wealth of standardized data allows for insights into nuclease function and its interplay with biology to engineer a therapeutic outcome

▪ Modular gRNA allow for controlling the core of the RNP medicine

▪ Cpf1 editing points to greater flexibility in developing CRISPR medicines

Poster : 7, 33 and 68

defining and characterizing the components of a crispr-cas9 … · crispr medicine guide + sequence...

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