Science - USA (2022-04-29)

and technical complexity (Fig. 1B), by sequen-
tially introducing mutations into the RB
pathway, then the MAPK pathway, and then
the telomerase regulation pathway through
precise editing of healthy human melanocytes.
We knocked out theCDKN2A(“C”, RB path-
way) by electroporating a genome-editing Cas9
RNP targeting theCDKN2Alocus (exon 2,
shared by both of its protein products, p16
and p14) ( 30 ). Small insertions and deletions
(indels) in the gene underwent positive selec-
tion in culture, reaching 90 to 95% mutated
allelefrequencybyday3and98to99%by
day 42 [Fig. 1C; mutated allele frequency
quantified as percent of alleles with an indel;

the two predominant alleles were a 79–and

53 – base pair (bp) deletions between the guides

of pairs 1 and 2 at 88 and 75%, respectively].

We next introduced the BRAF V600E mutation

(“B”, MAPK pathway) into C-edited melano-

cytes by codelivering Cas9 RNP targetingBRAF

exon 15 and a homologous DNA donor encod-

ing the V600E mutation ( 30 ). Recombinant

adeno-associated virus (AAV) was used to

deliver the DNA donor to overcome the low

editing efficiency of the single-stranded oligo-

deoxynucleotide donors (<0.25% at day 6;

table S1) ( 31 ). TheBRAFV600E allele fre-

quency increased from 6% at day 3 to 97%

at day 155 in culture (Fig. 1D, reflecting a

homozygous population and likely indicat-

ing that, in this context, two V600E alleles

provide a greater fitness advantage than one

allele). Finally, we introduced theTERT-124C>T

promoter mutation (“T”, telomerase regulation)

into“CB”melanocytes, codelivering a Cas9 RNP

targetingTERTexon 1 and a homologous DNA

donor encodingTERT-124C>T. The frequency

of the -124C>TTERTmutated allele increased

from 3 to 5% in the first 30 days in culture to

45% by day 75 and stayed at 41 to 50% for

more than 300 days of continuous culture (Fig.

1E; ascertained as predominantly heterozygous

with a small subpopulation of homozygous

cells) ( 30 ). Although the more common mutation

Hodiset al.,Science 376 , eabi8175 (2022) 29 April 2022 2 of 14

C DE

Mutant allele (%)

Log

10

(mRNA copies)

per 5 ng Total RNA

100

50

0

0 20

Days after genome editing Days after genome editing Days after genome editing

40 0 50 100 150 0 100 200 300

PTEN (“P”) Exon 5 indels TP53 (“3”) Exon 5 indels APC (“A”) Exon 17 indels

CDKN2A (“C”) Exon 2 indels BRAF (“B”) V600E TERT (“T”) –124 C>T prom. mut.

Guide pair 1

Guide pair 2

Non-targeting guide pair

#

#

Guide + donor

Guide + donor

Non-targeting guide + donor

Stopped dividing

I

0 10203040

H

Mutant allele (%)

0 10 20 30 40

G

TP53 (“3”) Exon 5 indels APC (“A”) Exon 17 indels

J K

100

50

0

0 20

Days after genome editing Days after genome editing Days after genome editing Days after genome editing Days after genome editing

40 60 80

Guide pair 1

Guide pair 2

Non-targeting guide

Guide pair 1

Guide pair 2

Non-targeting guide

Guide pair 1

Guide pair 2

Non-targeting guide

Guide pair 1

Guide pair 2

Non-targeting guide

Guide pair 1

Guide pair 2

Non-targeting guide

0 10 20 30 40 0 10 20 30 40

A B

Primary human

melanocytes

High-purity

mutant

population

Repeat process

to introduce each

additional mutation

Mutant cell

Cell culture

1–4 months

Natural selection

Electroporation

Cas9 RNP

AAV DNA donor (opt.)

F

0

1

2

3

4

5

6

7

TERT ACTB

( actin)

CB

CBT

*

WT C

CBT CBTP CBT CBT3 CBT CBTA CBTP CBTP3 CBTP CBTPA

C CB CB CBT

WT C CB CBT CBTP

CBT3

CBTA

CBTP3

CBTPA

CDKN2A–/–

BRAF V600E

CDKN2A–/–

TERT –124C>T

BRAF V600E –124C>TV600E

–124C>T

V600E

–124C>T

V600E

–124C>T

–124C>T V600E

V600E

CDKN2A–/–

TP53–/–

TERT

BRAF

CDKN2A–/–

PTEN–/–

TERT

BRAF

CDKN2A–/–

APC–/–

TERT

BRAF

CDKN2A–/–

PTEN–/–

TERT

BRAF

CDKN2A–/–

TP53–/–

PTEN–/–

TERT

BRAF

CDKN2A–/–

APC–/–

RB MAPK

PI3K / Akt

Wnt

p53

p53

Wnt

Primary

human

melanocyte

Telomerase

Fig. 1. Fitness advantage of cancer-driving mutations enables the
creation of a progressive series of genome-edited human cancer models.
(A) Experimental approach for introducing sequential melanoma mutations
into the genomes of primary human melanocytes with CRISPR-Cas9. RNP,
ribonucleoprotein; AAV, adeno-associated virus. (B) Editing tree showing the
nine isogenic models of melanoma generated (boxes), the perturbed genes in
each model (white boxes), the genotype abbreviation (beige boxes), and the
molecular pathway dysregulated by the most recent genome edit (red text).
(CtoE) Sequential introduction of first three mutations by CRISPR-Cas9
genome editing of wild-type (WT) melanocytes. (C) First mutation:CDKN2A
(“C”). (D) Second mutation:BRAF(“B”). (E) Third mutation:TERT(“T”).
TERTediting confers replicative immortality to CB melanocytes. Allele

frequencies of each engineered mutation (yaxis) shown over time (xaxis).

#, measurement of allele frequency discontinued because of cell senescence.

(F) Addition of the -124C>TTERTpromoter mutation activatesTERTexpression.

Mean of log 10 number ofTERTandb-actin (ACTB) transcripts (yaxis)

measured by qPCR in CB (black) and CBT (red) cells. Error bars, SD.n= 3.

*,P< 0.01, one-tailed, one-sample Student’sttest. (GtoI) Introduction

of fourth mutation into CBT melanocytes. (G) Allele frequencies of knockout

ofPTEN(“P”), (H) knockout ofTP53(“ 3 ”), and (I) knockout ofAPC(“A”).

(JandK) Introduction of fifth mutations into CBTP melanocytes (J) Allele

frequency of knockout ofPTENand (K) knockout ofTP53. Allele frequencies

(yaxis) shown over time (xaxis), as assessed by indels in the respective

loci in genomic DNA.

RESEARCH | RESEARCH ARTICLE