editasmedicine.com © 2018 Editas Medicine 1

Methods for Gene Editing Measurement 

and Off-Target Discovery

NIST-FDA Genome Editing Workshop

April 23, 2018 Gaithersburg, MD

Christopher Wilson, Ph.D.

Senior Director, Lead Discovery
© 2018 Editas Medicine 2

Disclosures for Chris Wilson 

▪ Employee and stock option holder of Editas Medicine
© 2018 Editas Medicine 3

Agenda

▪ LCA10 and CEP290 background

▪ UDiTaS

– to measure large deletions and inversions at the CEP290 editing site

– to measure translocations

– development of accuracy standards for structural changes

▪ Specificity approaches

– Digenome

– Statistical framework for describing off-target verification
© 2018 Editas Medicine 4

Leber Congenital Amaurosis Type 10

▪ Infantile-onset of poor vision, 

nystagmus, and a flat 

electroretinogram1

▪ Caused by autosomal recessive 

mutations in the CEP290 gene at 

chromosome12q21.322

▪ ~85% of LCA10 patients from northwest 

Europe have a “IVS26” mutation in 

intron 26, c.2991+1655A>G1-7

▪ CEP290 is present in the connecting 

cilium and is important for ciliogenesis, 

ciliary trafficking, and outer segment 

function and structure8

1Weleber RG, 2013 LCA Gene Reviews; 2den Hollander AI, Koenekoop RK, Am J Hum Genet 2006;79:556; 3Stone EM, Am J Ophthalmol

2007;144:791; 4CEP290_database 2017; 5Perrault I, Hum Mutat 2007;28:416; 6Vallespin E, IOVS 2007;48:5653; 7Simonelli F, IOVS 

2008;48:4284; 8Rachel RA, Cilia 2012;1:22

Outer segment Inner segment

Connecting 

cilium

CEP290

Discs

Outer segment

Degenerated

discs

Inner segment

low / no 

CEP290

WT Photoreceptor LCA10 Photoreceptor
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Gene Editing to Repair CEP290 Splicing Defect

DNA 

mRNA 

Protein

Exon 26 * Exon 27

IVS26

Exon 26 Exon 27

323 64

Deletion

Inversion

*

IVS26

CEP290 with IVS26 mutation CEP290 corrected with EDIT-101

Exon 26 X Exon 27 Exon 26 Exon 27

Transcription

Translation

p.Cys998X

Prematurely truncated 

and non-functional

Full length,

functional 

CEP290

NIST-FDA NIST-FDA Genome Editing Workshop | April 23, 2018 | Gaithersburg, MD | Christopher Wilson
NIST-FDA NIST-FDA Genome Editing Workshop | April 23, 2018 | Gaithersburg, MD | Christopher Wilson © 2018 Editas Medicine 6

Challenges with PCR-NGS assays 

when making multiple edits

1.1 kb • 3 PCR assays needed to measure 

editing at CEP290 intron 26 locus

• Even with rigorous standards it is 

difficult to cross compare assays

• Another set need for inversions

• ddPCR sufficient to measure the 

deletion but unable to distinguish 

inversions from wild type locus

Productive 

deletion

1.1 kb

Productive 

Inversion 1.1 kb

Productive 

deletion
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Custom Transposon

 UMI Barcode

 Pooling Barcode

 i5

5’

5’

3’

3’ ddC

Tn5* Tn5*

a

Custom 

Transposon

Genomic DNA from edited 

cells

+

Tagmentation

~ 2 kb fragments

PCR Round 1

i5 primer

Sequence 

specific primer

PCR Round 2

i7

i5 BC UMI BC i7

Sequencing Library

Editing event

Barcode

b

2

nd round primer

Uni-Directional Targeted Sequencing UDiTaS*

An NGS method for measuring junctions (and indels!)

* Giannoukos, et al., “UDiTaSTM, a genome editing detection method for 

indels and genome rearrangements”, BMC Genomics, 2018 19:212

c

Detection of editing

Detection of large deletions

Detection of inversions

CEP290 locus

UDiTaS primer

UMI

UMI

UMI

UDiTaS primer

UDiTaS primer

New junction

1,176 bp

guide 64 guide 323

New junction
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UDiTaS finds novel structural changes and is robust

U-2 OS cells nucleofected with 

plasmids expressing

SaCas9 + gRNA64 + gRNA323

gDNA from stable HEK293 line with 

deletion and inversion mixed with 

HEK293 gDNA at various ratios
© 2018 Editas Medicine 9

PK/PD relationship: On-target Editing Correlates with 

Transgene Expression by EDIT-101 in HuCEP290 KI Mice

UDiTaS in action: hundreds of samples 

from mouse pharmacology experiments

105 106 107 108 109 1010

0

10

20

30

40

50

60

70

Cas9 mRNA (Molecule/ug RNA)

Total Editing Rates (%)

Measured 

by UDiTaS
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UDiTaS can measure translocations

P

TRAC – chr14

Q

P

B2M – chr 15

Q

Schematic Type 1 (%) 2 (%)

No edits 14.87 n/a

Indels 82.35 n/a

Dicentric n/a n/a

Acentric 0.001 n/a

No edits n/a 3.89

Indels n/a 91.38

Dicentric n/a 0.42

Acentric n/a n/a

Balanced n/a n/a

Balanced 2.04 2.63

Acentric 0.66 n/a

Dicentric n/a 1.62

1

2

Q

Q

Q P

Q P

P

P

Q P

Q P

Q

Q

P

P

Q P

Q P

Q

Q

P

P

CD4+ human primary T cell

nucleofected with 2 RNPs:

• 10 possible outcomes

• 7 measurable with 

primers 1 and 2

• All 7 events detected
© 2018 Editas Medicine 11

Accuracy of translocation measurements

▪ Created series of 6 plasmid standards 

at the predicted TRAC-B2M 

translocation adding SNPs every ~10bp

▪ Individually dilute each plasmid ~3,000 

molecules to 3e6 molecules (3 logs)

▪ Spike into mouse genomic DNA

▪ Run UDiTaS and AMP-Seq 

▪ Demonstrates high accuracy and 

linearity of UDiTaS

B2M primer (#2)

TRAC primer (#1)

y = 6.06 x

0.84

y = 142.78 x

0.42

y = 4.49 x

0.86

y = 73.27 x

0.47

* * * * * * * * TRAC B2M

* * * * * * * * TRAC B2M

NIST-FDA NIST-FDA Genome Editing Workshop | April 23, 2018 | Gaithersburg, MD | Christopher Wilson
© 2018 Editas Medicine 12

Editas approach to editing specificity

Gene editing agent(s)

Discovery

Phase

Biochemical

Digenome-Seq

Cellular

GUIDE-Seq

In silico 

“closest matches”

Targeted Sequencing Assay Panels

in cells as relevant as possible

Off-target sites 

Verification

Phase

Risk assessment 

and mitigation 
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Digenome-Seq with Lead Guides (64 and 323)

CEP290 Guide 323 

on-target example

+

genomic DNA

16 

hours WGS Identify overabundant start sites Align & 

analyze

CAS9 + Guide

Count of sites

Includes 

on-target site
© 2018 Editas Medicine 14

Statistical framework for sensitivity calculations 

• Similar to small molecule 

safety profiling

• Off-target measurements 

needs to include the limit 

of detection

• Express no detected offtargets as <LLoD; eg: 

“Editing at chr1:124245 

is <0.1%”

• Main determinants are:

• Read count

• Input DNA amount

Binomial sampling distribution in NGS 

as part of an LLoD determination
© 2018 Editas Medicine 15

Summary

▪ LCA10 and CEP290 background

▪ UDiTaS

– to measure large deletions and inversions at the CEP290 editing site

– to measure translocations

– development of accuracy standards for structural changes

▪ Specificity approaches

– Digenome

– Statistical framework for describing off-target verification (and lack of off-target 

measurement)
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Thank you.