Science - USA (2021-07-09)

Wagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 9 of 13

Control Chr21 miRNAs

FSC-A

SSC-A

CD34-BV421

CD117-APC

N-FL N-FL

CD45+ Cells:

Out of CD45+:

ABSort LT-HSCs

from N-FL

Lentiviral transduction of

miR-99a, miR-125b-2 and miR-155

(Chr21 miRNAs)

Xenotransplantation

(12 weeks)

Differentiation analysis:

flow cytometry

• Megakaryocytic


• Erythroid


• Myeloid


• Lymphoid

SFFV mOrange

CD

F H

J

E

I K

GSort LT-HSCs

from T21-FL

Xenotransplantation

(20 weeks)

Electroporation of RNPs

Cas9

GATA1s

STAG2ko

Cas9

miR-99a KO

miR-125b-2 KO

miR-155 KO

Leukemic analysis:

flow cytometry + genotyping

+

• Blast

24h

T21-FL

Control KO

GATA1s

T21-FL

Control KO

GATA1s/STAG2ko

T21-FL

Chr21 miRNAs KO

GATA1s

FSC-A

SSC-A

CD34-BV421

CD117-PE

CD45+ Blast:

T21-FL

Chr21 miRNAs KO

GATA1s/STAG2ko

Out of CD45+:

ControlGAT

A1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

% CD45+ Engraftment

Control

KO

Chr21

miRNAs KO

Control

Chr21 miRNAs

Control

Chr21 miRNAs

0

20

40

60

80

100

B-Lymphoid-

Myeloid-

Erythroid-

T-cell-

Megakaryocytic-

% Lineage marker distribution

Marker

Bone

marrowSpleen

N-FL

****

***

Control

Chr21 miRNAs

Control

Chr21 miRNAs

0

20

40

60

80

100

% CD45+mOrange+ Engraftment

Bone

marrowSpleen

N-FL

**** ****

ControlGATA1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

80

% CD117+CD45+ (out of blast gate)

Control

KO

Chr21

miRNAs KO

ControlGATA1s

GATA1s/STAG2ko

ControlGATA1s

GATA1s/STAG2ko

0

20

40

60

80

100

% Human cells

Unknown

Blast

Myeloid cells

(not blast-like)

Erythroid

precursors

Control

KO

Lymphocytes

Chr21

miRNAs KO

Control

Chr21 miRNAs

0

20

40

60

80

100

% Human cells

N-FL

Unknown

Erythroid

precursors

Lymphocytes

Myeloid cells

(not blast-like)

Blast

miR-99a-5p

miR-125b-2-5p

miR-155-5p

let-7c-5p

1

2

4

8

16

32

Relative expression to N-FL

(normalized by

RNU48

)

T21-FL

LT-HSC

* **

Fig. 5. Chr21 miRNAs predispose to preleukemia.(A) Relative expression of
Chr21 miRNAs in T21-FL LT-HSCs compared with N-FL as measured with reverse-
transcription quantitative PCR (n= 3 replicates per condition). (B) Overview
of lentiviral mediated overexpression of Chr21 miRNAs in N-FL LT-HSCs used for
primary xenotransplantation into NSG and NSGW41 mice. (C) Engraftment of
transduced control and Chr21 miRNAs in N-FL LT-HSCs transplanted into NSG
mice (only mice with >1% CD45+cells in BM were analyzed,n= 9 or 10 mice per
condition). (D) Lineage marker distribution based on cell surface markers of
engrafted NSG mice in (C). (E) Quantification of cell morphology of human cells
prepared by using cytospin in grafts described in (C) (n= 400 cells per
condition). (F) Flow cytometry plots out of CD45+cells in primary xenografts
described in (C). (G) Experimental overview of sorting T21-FL LT-HSCs for
CRISPR/Cas9 editing with miR-99a, miR-125b-2, and miR-155 gRNAs and

subsequently with GATA1s and STAG2 gRNAs for primary xenotransplantation

into NSG mice. (H) Engraftment of control knockout (KO) and Chr21 miRNAs

KO in T21-FL LT-HSCs transplanted into NSG mice. Each subgroup was

additionally edited with control, GATA1s, and GATA1s/STAG2 gRNAs (only mice

with >1% CD45+cells in BM and >90% CRISPR/Cas9 efficiency are depicted,

n= 5 to 10 mice per condition). (I) Quantification of cell morphology of

human cells prepared by using cytospin in transplanted NSG mice described

in (H) (n= 400 cells per condition). (J) Flow cytometry plots showing blast

populations out of CD45+cells in primary xenografts described in (H).

(K) Quantification of CD117+CD45+blasts of transplanted NSG mice described

in (H) (§§§ indicate significance in relation to GATA1s control KO). Unpaired

Student’sttest: *P< 0.05; **P< 0.01; ***/§§§P< 0.001; ****P< 0.0001;

error bars indicate standard deviation.

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