Developing Meganucleases for 

Clinical Applications

Derek Jantz PhD, Chief Scientific Officer 
Meganucleases

Meganucleases are engineered variants of the I-CreI homing endonuclease from 

Chlamydomonas reinhardtii

• I-CreI targets the insertion of a Group I intron into the 23S rRNA gene

• The I-CreI target site is 22bp; cleavage produces a pair of 4bp 3’ overhangs

• Meganucleases are small (310 aa) and monomeric

• Meganucleases recognizes DNA through direct amino acid side-chain-to-base 

contacts that can be changed to confer novel binding specificities.

5’-CAAACTGTCGTGA

3’-GTTTGACAG

GACGTTTTG-3’

CACTCTGCAAAAC-5’
GCAAACGTCGTGAGACAGTTTCG

wild-type I-CreI target site in Chlamydomonas

TRC 1-2x.87EE: a meganuclease for T cell editing

TRC 1-2x.87EE

TGGCCTGGAGCAACAAATCTGA

target site in human TRAC gene

I-CreI

• TRC 1-2x.87EE is a single-chain meganuclease that recognizes Exon 1 of the human TRAC gene

Linker
“One step” allogeneic CAR-T cells

T cell Receptor Alpha Constant (TRAC) locus

Exon 1 Exon 2 Exon 3 Exon 4

CAR

AAV6

LHA JeT CD19-N6-z CAR RHA

Meganuclease mRNA

T cell Receptor Alpha Constant (TRAC) locus

Exon 1 Exon 2 Exon 3 Exon 4

Electroporate

Homologous

Recombination

~70% yield

JeT
TRC 1-2x.87EE cuts off-target frequently

44 confirmed off-target sites

2 171834829 60 T A G T G T A A 7

4 113389218 59 G T T C C A 5

7 121204561 59 T A G T G T A A 7

15 93059767 55 T A G T G T A A 7

4 46296485 54 G A T G C C A 5

3 124472302 53 T A G T G T A A 7

3 190642718 52 A G T G T A A 6

2 219039632 52 T A G T G T A A 7

3 170536265 51 T A G T G T A A 7

6 128649180 51 G A T T C G 6

X 126002084 50 T A A G G T G T A A 7

7 15547932 49 G A T T A C A A A 8

17 73253472 49 T A G T G T A A 7

X 85564817 48 T T C C C A 5

1 53894790 47 T A G T G T A A 7

17 32515115 47 T A G T G T A A 7

6 110177848 45 T A G T G T A A 7

9 80800844 45 T A G T G T A A 7

21 44221868 43 T A G T G T A A 7

2 45262483 43 T A G T G T A A 7

4 88857981 43 T A G T G T A A 7

6 102744289 43 T A G T G T A A 7

10 5454314 43 T T G A G T C A 6

14 23016633 1956 T G G C C T G G A G C A A C A A A T C T G A

A 5 3 3 4 8 2 1 0 93 5 63 90 89 13 92 12 32 4 5 62 77 93

T 88 3 3 4 63 96 1 8 2 3 24 4 2 2 1 1 63 16 61 22 2 3

G 4 92 93 18 1 1 97 91 3 90 6 5 3 2 7 70 1 62 1 4 20 2

C 2 1 2 73 28 0 1 1 2 1 7 1 6 83 0 17 4 18 34 12 1 2

Chr BP Reads Mismatches

20 56622815 730 C T T C A G G C 6

3 61066689 401 C A G A G C 4

11 112420069 383 G A G 1

8 77742673 309 T T A T 2

4 2739527 300 G T C A 3

4 175903457 232 T C C C A 4

1 36350764 231 G A A G G G G T C 7

4 46641994 171 G T C C 3

2 62675694 164 G A T C A G G A 6

X 30201957 149 T T C G T G A 6

TRC 1-2x.87 EE

Repeated sequence

Off-2

Off-1

Off-target sites cut 

with high frequency
A Second-Generation Nuclease Was Produced

TRC 1-2x.87EE 

(Generation 1)

TGGCCTGGAGCAACAAATCTGA

target site in human TRAC gene

TRC 1-2L.1592 

(Generation 2)

TGGCCTGGAGCAACAAATCTGA

target site in human TRAC gene

H40

K28

V40

Linker Linker
Off-2 target:

High Frequency

TRC 1-2L.1592 does not cut Off-1 or Off-2 in a reporter assay

0.00

0.20

0.40

0.60

0.80

1.00

1.20

10X 1X 10X 5X 10X 10X

mock TRC 1-2x.87EE TRC 1-2L.1592 cntrl

day 2

day 5

day 7

0.00

0.20

0.40

0.60

0.80

1.00

1.20

10X 1X 10X 5X 10X 10X

mock TRC 1-2x.87EE TRC 1-2L.1592 CHO 23-24

day 2

day 5

day 7

Off-1 target: 

Repeat

Normalized editing efficiency Normalized editing efficiency 
Genome-Wide Analysis

1. In silico site prediction

2. GUIDE-seq

3. Oligo Capture

Targeted Indel Analysis

Screen all putative off-target sites 

(2,000-10,000 reads each) 

Confirmation

>10,000 reads, 

triplicate

Breadth of Coverage

Depth of Coverage

TRC 1-2L.1592 off-target analysis
Genome-Wide Analysis 1: in silico site prediction

All sites in the genome <4bp different from the intended target site (COSMID)

site # Chr Pos Triplicate? Indel Frequency (%)

site 0 14 23016633 T G G C C T G G A G C A A C A A A T C T G A Y 61.35

site 1 19 50167592 A C C N N.S

site 2 2 121248255 C A C N N.S

site 3 2 168041009 G A A N N.S

site 4 7 124214889 G T A N N.S

site 5 11 579363 C A T N N.S

site 6 8 1220286 T G C N N.S

site 7 X 90307430 A A T N N.S

site 8 11 112420069 G A G Y N.S

site 9 6 146944540 T T A N N.S

site 10 X 130371540 A A T N N.S

site 11 2 192882479 A T T N N.S

site 12 18 52058914 C G C N N.S

site 13 11 65311098 C G G N N.S

site 14 17 70963906 T C C Y 0.23

site 15 1 30736654 A A N N.S

Sequence

N.S.: not significant (<1 STDEV over mock)
Genome-Wide Analysis 2: GUIDE-seq

Standard protocol and software modified to remove requirement for PAM sequence

site # Chr Pos Triplicate? Indel Frequency (%)

site 0 14 23016633 T G G C C T G G A G C A A C A A A T C T G A Y 61.35

site 200 3 45249461 G A G A T C C N N.S

site 201 4 149232383 T T T T T T T N N.S

Sequence

N.S.: not significant (<1 STDEV over mock)
Sticky-end dsODN

dsDNA in cell genome

dsODN tag integration in live cells

Genomic DNA isolation

Random shearing

End Repair, adapter ligation

dsODN specific amplification

NGS library prep and sequencing

Molecular index

Adapter Oligo tag

Merge R1 

and R2

Filter for reads that 

have complete 

adapters and oligos

Trim adapter 

and oligo 

sequence

Pre-Processing

Align to 

human 

genome

Remove 

PCR 

duplicates

Identify 

off-target 

sites

Off-target identification

Filter using 

mock 

sample

Raw data 

(fastq)

Off target 

tables

Oligo Capture

(GUIDE-seq differences highlighted in blue)

Adapted from Tsai, et al. Nature Biotech 2015, 33(2), 187-197

dsODN

integration

Blunt Overhangs

cassette captured in TRAC gene

Results not filtered based 

on similarity to intended 

target site
Genome-Wide Analysis 3: Oligo Capture

site # Chr Pos Triplicate? Indel Frequency (%)

site 0 14 23016633 T G G C C T G G A G C A A C A A A T C T G A Y 61.35

site 8 11 112420069 G A G Y N.S

site 14 17 70963906 T C C Y 0.23

site 43 7 45886383 G G A T A C Y 0.44

site 46 11 117707291 T A C C T Y 0.85

site 79 8 70953326 G T A C C Y 2.83

site 80 15 91730805 T T G T A C T Y 0.45

site 81 6 134362399 G T A G G G A A T N N.S

site 83 20 51802795 T A A T T G A C T N N.S

site 84 2 85864762 A A C A C T T C A C N N.S

site 85 5 75148730 A A T T T T A A N N.S

site 86 14 96013249 A T G T T G T G C N N.S

site 89 8 68898594 A A G G C C A T N N.S

site 92 22 48584419 A A A A G T A C C T N N.S

site 93 2 9176023 A A G A T T T C T C T N N.S

site 94 4 183627898 T T T A T C T G C A Y N.S

site 95 6 86300063 A T T T T G G A A A T N N.S

site 97 11 67957795 A A G T T G T C G C T N N.S

site 98 10 94357492 T A C C G G G G G Y N.S

site 100 2 200426071 T T A T T T G T G A C N N.S

site 101 7 102266445 A T G A T G A C G T N N.S

site 102 12 133336771 A A A T C C C C A A T N N.S

site 103 1 91577468 A T A C T A G C A N N.S

site 105 2 207017087 A T A T T T T A A A Y N.S

Sequence

(47 total candidate sites identified)
Off-target site locations

• No sites in exons

• Three sites in non-coding regions of genes

• No known oncogenes

• No known association with relevant pathology

Zinc transporter

Na,K-ATPase regulation

Uncharacterized secretory role

Site # % indel Location

site 14 0.23 Intron 4 of SLC39A11 , >10 kb from the nearest exon

site 43 0.44 >10 kb from closest known gene transcript

site 46 0.85 3' UTR of FXYD6, 3225 bp from the nearest exon 

site 79 2.83 >10 kb from closest known gene transcript

site 80 0.45 Intron 1 of SV2B , >10kb from the nearest exon
Editing safety studies

Study TRC 1-2x.87EE (gen 1) TRC 1-2L.1592 (gen 2) 

Karyotype N.D. Negative

IL-2 Independent Growth Negative Negative

NSG Mouse Tumorigenesis Negative Negative
x.87EE vs. L.1592 

x.87EE

L.1592

70.7%

28.1%

76.7%

22.4%

CD3

CAR

L.1592

Knock-In Efficiency

x.87EE

L.1592

CD4: 43.6%

CD8: 54.8%

CD4: 49.4%

CD8: 49.2%

CD8

CD4

CD4:CD8 Ratio

C 1 -2 x .8 7 E E + A A V

R C 1 -2 L .1 4 6 9 + A A V

R C 1 -2 L .1 5 9 2 + A A VR C 1 -2 L .1 7 7 5 + A A V

R C 1 -2 L .1 7 6 2 + A A V

R C 1 -2 L .1 8 4 3 + A A V

0

2 0

4 0

6 0

8 0

1 0 0

% o f s u b s e t

N aive C e n tra 

E ffe c to 

C 1 -2 x .8 7 E E + A A V

R C 1 -2 L .1 4 6 9 + A A V

R C 1 -2 L .1 5 9 2 + A A V

R C 1 -2 L .1 7 7 5 + A A V

R C 1 -2 L .1 7 6 2 + A A V

R C 1 -2 L .1 8 4 3 + A A V

0

2 0

4 0

6 0

8 0

1 0 0

% o f s u b s e t

N aive C e n tra l M e m o ry E ffe c to r M e m o r

T R C 1 -2 x .8 7 E E + A A V

T R C 1 -2 L .1 4 6 9 + A A VT R C 1 -2 L .1 5 9 2 + A A VT R C 1 -2 L .1 7 7 5 + A A VT R C 1 -2 L .1 7 6 2 + A A VT R C 1 -2 L .1 8 4 3 + A A V

0

2 0

4 0

6 0

8 0

1 0 0

% o f s u b s e t

N aive C e n tra l M e m o ry E ffe c to r M e m o ry

Phenotype Viability

0

10

20

30

40

50

60

70

80

90

100

Day 3 Day 8 Day 13

TRC 1-2x.87EE

TRC 1-2L.1592
x.87EE vs. L.1592

8 7 E E 1 5 9 2 R a j i o n l y

0

1 0 0 0 0 0

2 0 0 0 0 0

3 0 0 0 0 0

R a j i 1 : 2 C A R T

T o t a l C A R T i n c u l t u r e

8 7 E E

1 5 9 2 R a j i o n l y

* * * *

* * * * p < 0 . 0 0 0 1

Cell expansion

8 7 E E 1 5 9 2 R a j i o n l y

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

R a j i 1 : 2 C D 1 9 +

T o t a l C D 1 9 + i n c u l t u r e

8 7 E E

1 5 9 2 R a j i o n l y

* p < 0 . 0 5

*

Target cell killing expansion

Enumerate CAR T cell and 

target number by flow 

cytometry

Co-culture CAR T cells

And targets at 

1 target: 2 CAR T cells

5 days
x.87EE vs. L.1592
Concluding remarks

• Standardization across editing platforms will be challenging.

• Off-targeting can manifest itself in subtle ways that are probably more likely to 

affect efficacy/performance than safety.

Regarding off-targeting:

“Try the best we can...”

-Ed Rebar (yesterday, at least 3 times)
Thank You!