Science - USA (2022-04-29)

occasionally became apparent at the injection
site before tissue harvest [7 of 12 injections;
Fig. 3D, (n=8), inset: slight uptick of black
curve by day 151; and fig. S13, no growth by
day 189 (n=4)]. These small melanocytic
tumors were malignant by dermatopathologic
evaluation (four of four tumors examined; fig.

S14 and table S8). Thus, in line with observations

in patient melanomas ( 18 ), melanocytes with

common melanoma mutations in the endogenous

loci ofCDKN2A,BRAF, andTERTdisplayed

phenotypic characteristics of early melanomas.

Three alternative quadruple-mutation

branches of the editing tree—CBT3, CBTA,

and CBTP—each had distinct effects on mela-

noma phenotypes. CBT3 cells did not produce

visible tumors over a period of ~60 days, al-

though by day 69 a few injection sites (3 of 16)

began to show small tumors (≤14 mm^3 ;Fig.3B:

slight uptick of red curve by day 69). By con-

trast, CBTA cells formed darkly pigmented,

Hodiset al.,Science 376 , eabi8175 (2022) 29 April 2022 5 of 14

Days after injection of cells

36

— CBTP —

111 68 151

— CBT —

151

CBTP3

68

CBTA

111

CBTPA

36

G

Lung metastases per section

400

600

200

0

H

Liver metastases per section

30

40

20

10

0

I

% of initial weight

125

100

150

150

100

50

75

0

Days after injection of cells

Days after injection of cells

36

— CBTP —

111 68 151

— CBT —

151

CBTP3

68

CBTA

111

CBTPA

36

B C

A

0

D

Tumor volume (mm

3 )

0 50

Days after injection of cells Days after injection of cells Days after injection of cells

100 150

0

5

10

50 100 150 0 50 100 150

500

250

# * *

* *

E F

Days after injection of cells Days after injection of cells

0 50 100 150 0 50 100 150

* *

## * *

CBT3, Guide 1 (n = 8)

CBT3, Guide 2 (n = 8)

CBT, Non-targeting guide (n = 4)

CBTA, Guide 1 (n = 8)

CBTA, Guide 2 (n = 8)

CBT, Non-targeting guide (n = 8)

CBTP, Guide 1 (n = 8)

CBTP, Guide 2 (n = 8)

CBT, Non-targeting guide (n = 8)

CBTP3, Guide 1 (n = 8)

CBTP3, Guide 2 (n = 8)

CBTP, Non-targeting guide (n = 8)

CBTPA, Guide 1 (n = 8)

CBTPA, Guide 2 (n = 8)

CBTP, Non-targeting guide (n = 8)

Day 36

CBTPA

Guide

1

CBTPA

Guide

2

CBTP

Non-targ.

guide

Day 68

CBTP3

Guide

1

CBTP3

Guide

2

CBTP

Non-targ.

guide

Day 151

CBTP

Guide

2

CBTP

Guide

1

CBT

Non-targ.

guide

Day 111

CBTA

Guide

1

CBTA

Guide

2

CBT

Non-targ.

guide

Day 69

CBT3

Guide

1

CBT3

Guide

2

CBT

Non-targ.

guide

Mouse experiment 1

Mouse experiment 2

Mouse experiment 3

Mouse experiment 4

Mouse experiment 1

Mouse experiment 2

Mouse experiment 3

Mouse experiment 4

0

5

60 70 120 140

10

Size Morphology

Mutant melanocytescultured in vitro into dermis of immunodeficient mouseInjection of cells of a single genotype Tumor growth Metastasis Weight

36–151 days

CBTPA (n = 8)

CBTP3 (n = 8)

CBTP (n = 8)

CBTA (n = 8)

CBT3 (n = 8)

CBT (n = 4)

#

##

Fig. 3. Mutation combinations confer diverse, disease-relevant phenotypes
in vivo.(A) Experimental approach to identify disease-relevant phenotypes
caused by engineered mutations in vivo. (BtoF) Primary tumor growth
of xenografted mutant melanocytes in NSG mice compared with CBT or
CBTP control parental cells (as shown) that received nontargeting Cas9 RNP:
(B) CBT3, (C) CBTA, (D) CBTP, (E) CBTP3, and (F) CBTPA cells. Top panels:
tumor size (cubic millimeters,yaxis) over time (days,xaxis) after two intradermal
injections, one in each flank. n, number of tumors. Bottom panels: representative
images of (shaved) mice harboring mutant cells as marked. A ruler with large,
numbered marks in centimeters is shown for scale. (GandH) Loss ofAPC
promotes frequent distant metastases. Average number of individual metastatic
foci per section (symbols) of lung (G) or liver (H) tissue in a histologic slide

(yaxis, counted manually) obtained from a single mouse injected with a

mutant cell line (genotype indicated by color) and collected after the indicated

number of days (xaxis). Each slide had an average of three lung sections

and two liver sections, each from a different lobe. (I) Injected CBTPA melanocytes

cause rapid weight loss in mice. Percent of initial mouse weight [yaxis,

determined after subtracting primary tumor weights (estimated at 1 g/cm^3 )

from measured mouse weight]) over time (xaxis, days). n, number of

mice. Data in (G and H) are from the four independent experiments in

(C to F). #, two CBTA mice, one from each guide group, were sacrificed for

histological inspection (C and I). ##, one CBTPA mouse was euthanized as a

result of primary tumor ulceration (F and I). *P< 0.01; ns, not significant;

two-tailed, two-sample StudentÕsttest.

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