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Towards predictable editing: influence of target sequence, cell type, and choice of type II nuclease on

desired repair outcomesLuis Barrera, Barrett Steinberg, Derek Cerchione, Christopher Wilson, Cecilia Cotta-Ramusino, Vic Myer, Hari Jayaram

Editas RNP lead discoveryFigure 1.

RNP for Spy and Sau Cas9

variants is delivered to

HEK293T and T-cells (T cell

data shown) and efficacy

determined by NGS

sequencing in dose

response mode.

Number of active guides

across locus for both Spy

Cas9 and Sa Cas9.

Indel profiles depend on cell type and gRNA locus

Figure 2. The indel spectrum of Cas9 edits across a

series of gRNA in T-cells. gRNA range from those that

result in a dominant indel to those that have a wider

spectrum of indels.

T-cells prefer deletions and HEK-293T cells insertions

Figure 4. Probability density of edit sizes in T-cells vs

HEK293T cells. The skewing of indel patterns under

equivalent conditions indicates a cell type dependence.

Targeted DNA cleavage by type IICRISPR nucleases has enabled avariety of genome editingapplications.

However, the apparently stochasticrepair of DNA breaks can create abroad range of edited sequences,providing a unique challenge shoulda specific edit be desired.

Here, we investigated thedistributions of insertions anddeletions following cleavage by Cas9from S. pyogenes (Sp) and S. aureus(Sa) when paired with different guideRNAs in primary human T-cells andHEK 293T cell line.

We quantified the degree to whichspecific editing conditions createhighly dominant alleles and observedsignificant variability. In addition, weobserved shifts in the types andfrequencies of repair outcomes,particularly when comparing editingin primary cells and cell lines.

The analysis of repair outcomedistributions provides opportunitiesfor improving models of non-homologous end joining andsuggests mechanistic hypotheses forhow factors inherent to the nucleaseactivity influence the repair process. Figure 3. Comparison of guides with dominant indels

between T-cells and HEK-293T cells. Key deletions

indicated by arrows show similarities and differences

between cell types.

Abstract

Dominance of the +1 edit shown by Spy Cas9

Question?

References:1. 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.

Given the wealth of CRISPR

nucleases, how do we pick

between them to engineer a

therapeutic edit?

HEK293 T cells

R1 R2 R2R1

Predominant common 1 bpinsertion

HEK-preferred7 bpdeletion

T-cell-preferred17 bpdeletion HEK293 T cells

R1 R2 R2R1

Inde

l typ

e

Position (relative to cut site):Indellength

Guide 1 Guide 2

More control Less control

Variant, gRNA sequence and cell type influence edit

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

0.00

0.01

0.02

0.03

0.04

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.03

0.04

0.00

0.01

0.02

0.03

0.04

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.03

0.000

0.005

0.010

0.015

0.020

0.025

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.03

0.04

0.00

0.01

0.02

0.03

0.00

0.01

0.02

0.00

0.01

0.02

0.03

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

−200 −150 −100 −50 0 50 −100 −50 0 50 −50 −25 0 25 50 −25 0 25 50

−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

Figure 5. Nuclease dependent staggered cutting possibly

leads to predominant +1 edit in Spy Cas9 over Sau Cas9.

*

ConclusiongRNA range from showing a

dominant edit to a large

spectrum of edits dependent on

CRISPR variant and cell type.

Understanding these

relationships can allow

streamlining of gRNA screening

and engineering efforts.

*

Guide 1 Guide 2 Guide 3

Guide 4 Guide 5 Guide 6

guides

T1 T3 T4 T5T2