J Hematol

Journal of Hematology, ISSN 1927-1212 print, 1927-1220 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Hematol and Elmer Press Inc

Journal website https://jh.elmerpub.com

Original Article

Volume 15, Number 2, April 2026, pages 108-128

Engraftment Outcome of CRISPR/Cas9-Edited Hematopoietic Stem Cells for Genetic Diseases: A Systematic Review and Meta-Analysis of Preclinical Evidence

↓ Figure 2. CRISPR-Cas9 gene editing negatively impacts the ability of hematopoietic stem and progenitor cells to engraft in vivo, as measured by fluorescent-activated cell sorting. (a) Analysis of bone marrow engraftment comprised a total of 68 datasets. Most estimates were negative (68%), with observed standardized mean differences ranging from –1.8391 to 0.7740. (b) Analysis of spleen engraftment comprised a total of 24 datasets. Most estimates were negative (88%), and the observed standardized mean differences varied from –1.8629 to 0.4730. (c) Analysis of peripheral blood engraftment comprised a total of 41 datasets. The majority of estimates (78%) were negative, with observed standardized mean differences ranging from –1.1051 to 0.7224. CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9.

↓ Figure 3. The in vivo engraftment potential of human CD34⁺ HSPCs is impacted by the gene knockout approach used for gene editing. (a) Analysis of bone marrow engraftment included 38 datasets. The majority of estimates (76%) were negative, with observed standardized mean differences ranging from –1.8391 to 0.7740. (b) Analysis of spleen engraftment comprised a total of 14 datasets, of which 93% of the estimates were negative, with the observed standardized mean differences ranging from –1.8629 to 0.2046. (c) Analysis of peripheral blood engraftment comprised a total of 33 datasets. Most estimates (85%) were negative, with the observed standardized mean differences ranging from –1.1051 to 0.7534. HSPCs: hematopoietic stem and progenitor cells.

↓ Figure 4. CRISPR-Cas9-mediated gene editing using RNP complex affects the in vivo engraftment of human HSPCs in mice model. (a) The analysis of bone marrow engraftment comprised 61 datasets that used the RNP version of the CRISPR-Cas9 system. The majority of estimates (72%), with observed standardized mean differences ranging from –1.8391 to 0.7740, were negative. (b) The study of peripheral blood engraftment included 60 datasets that used the RNP version of the CRISPR-Cas9 technology. A significant proportion of estimates (77%), with observed standardized mean differences ranging from –1.1051 to 0.7534, were negative. (c) Twenty-one datasets in all that used the RNP version of the CRISPR-Cas9 technology were analyzed for spleen engraftment. The observed standardized mean differences were mostly negative (95%), with a range of –1.8629 to 0.2046. (d)The investigation of peripheral blood engraftment comprised a total of eight datasets that used the CRISPR-Cas9 system in RNA form. The majority of estimates (88%), with observed standardized mean differences ranging from –0.1343 to 0.4505, were positive. CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HSPCs: hematopoietic stem and progenitor cells; RNP: ribonucleoprotein complex.

↓ Figure 5. The genetic background of NSGS and NSG Inbred mice influences the engraftment of gene-edited hHSPCs in the spleen, bone marrow, and peripheral blood. (a) The analysis of bone marrow engraftment comprised 38 datasets that used the NSG strain of mice model. The majority of estimates (74%), with observed standardized mean differences ranging from –1.8391 to 0.5537, were negative. (b) Analysis of spleen engraftment was conducted on 11 datasets that used the NSG strain of mice model. All estimations were negative (100%), and the observed standardized mean differences varied from –1.8629 to -0.1399. (c) The analysis comprised five datasets on peripheral blood engraftment using the NSGS mouse strain model. The observed standardized mean differences varied from –0.5092 to 0.7534, with 60% of the estimations being positive. hHSPCs: human hematopoietic stem and progenitor cells.

↓ Figure 6. Utilizing the tail vein, intraperitoneal, and retro-orbital routes to deliver cells compromises the engraftment efficiency of gene-edited hHSPCs in the spleen and peripheral blood. The analysis of spleen engraftment comprised (a) eight datasets on spleen engraftment that used the tail vein as the delivery route. The observed standardized mean differences varied from –1.8629 to 0.2046, with 88% of estimates being negative. (b) Ten datasets that used retro-orbital administration. The observed standardized mean differences varied from –0.8680 to 0.2357, with 90% of the estimations being negative. (c) A total of four datasets that used intraperitoneal as the delivery method. All estimations were negative (100%), and the observed standardized mean differences varied from –1.4638 to -0.5878. In order to analyze peripheral blood engraftment (d), 45 datasets that used tail vein injection were considered. A significant portion of estimates (73%), with observed standardized mean differences ranging from –0.9161 to 0.4505, were negative. (e) A total of four datasets with intraperitoneal delivery were included. All estimations were negative (100%), and the observed standardized mean differences varied from –1.1051 to –0.7621. hHSPCs: human hematopoietic stem and progenitor cells.

↓ Figure 7. The engraftment capacity of cells is affected by CRISPR-Cas9-based gene editing of peripheral blood-derived HSPCs and hemoglobinopathy-related genes. The investigation of HSPCs obtained from peripheral blood in (a) bone marrow engraftment included 58 datasets in total. With 72% of estimations being negative, the observed standardized mean differences varied from –1.8391 to 0.7740. (b) Peripheral blood engraftment included a total of 60 datasets. The observed standardized mean differences ranged from –1.1051 to 0.7534, with 70% of estimations being negative. To examine the editing effect of hemoglobinopathy-related genes (c) on peripheral blood engraftment, 40 datasets were analyzed. The majority of estimates (78%), with observed standardized mean differences ranging from –1.1051 to 0.7224, were negative. (d) For spleen engraftment, data from 13 datasets were utilized. Most estimates (85%) were negative, and the observed standardized mean differences varied from –1.4431 to 0.2338. CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HSPCs: hematopoietic stem and progenitor cells.

↓ **Table 1.** Characteristic Summary of the Preclinical Studies Included in the Metanalysis
Author and year	Goal of gene editing	Cas9 delivery system	Donor template delivery system	Targeted gene	Tissue source of hHSPCs	Mice strain	Mode of transplantation	Number of cells injected	Follow-up duration after transplantation
hHSPCs: human hematopoietic stem and progenitor cells; mRNA: messenger ribonucleic acid; gRNA: guide RNA; RNP: ribonucleoprotein complex; AAV6: adeno-associated virus type 6; ssODN: single-stranded oligodeoxynucleotides; PB: peripheral blood; BM: bone marrow; UCB: umbilical cord blood; CCR5: C-C chemokine receptor type 5; TET2: Tet methylcytosine deoxygenase 2; CYBB: cytochrome b-245 beta chain; HBB: hemoglobin subunit beta; CD: cluster of differentiation; HBG: hemoglobin subunit gamma; ITGB2: integrin beta subunit 2; ELANE: neutrophil elastase; FXN: frataxin; FOXP3: forkhead box P3; MAGT1: magnesium transporter 1; BCL11: B-cell lymphoma/leukemia 11; PKLR: pyruvate kinase L/R; PRR: putative repressor region; WAS: Wiskott Aldrich syndrome; IL2RG: interleukin 2 receptor subunit gamma; BTK: Bruton’s tyrosine kinase; NSG: non-obese diabetic (NOD) Scid gamma.
Xu et al, 2017 [19]	Knockout	Plasmid	No donor template	CCR5	Fetal liver	NSG	Intrahepatic	1 × 10⁶	12 weeks
Tothova et al, 2017 [20]	Knockout	Plasmid	No donor template	TET2/Cohesin	PB	NSGS	Retro-orbital	0.2–0.5 × 10⁶	22 weeks
De Ravin et al, 2017 [21]	Knock-in	mRNA/gRNA	ssODN	CYBB	PB	NSG	Tail vein	1–3 × 10⁶	20 weeks
Yen et al, 2018 [22]	Knockout	RNP	No donor template	HBB	BM	NSG	Tail vein	0.7 × 10⁶	12–20 weeks
Kim et al, 2018 [23]	Knockout	RNP	No donor template	CD33	PB	NSG	Tail vein	0.1–0.5 × 10⁶	16 weeks
Pattabhi et al, 2019 [24]	Knock-in	RNP	AAV6/ssODN	HBB	PB	NBSGW	Tail vein	2 × 10⁶	14 weeks
Borot et al, 2019 [25]	Knockout	RNP	No donor template	CD33	BM	NSGSGM3	Tail vein	0.5–1 × 10⁶	9–21 weeks
Romero et al, 2019 [26]	Knock-in	RNP	AAV6/ssODN	HBB	PB	NSG	Retro-orbital	1–1.3 × 10⁶	16 weeks
Metais et al, 2019 [27]	Knockout	RNP	No donor template	HBG1/HBG2	PB	NBSGW	Tail vein	1 × 10⁶	16–17 weeks
Bloomer et al, 2021 [28]	Knock-in	RNP	AAV6	ITGB2	PB	NSG	Tail vein	0.3 × 10⁶	18 weeks
Yudovich et al, 2020 [29]	Knockout	RNP	No donor template	CD45	UCB	NSG	Tail vein	0.1 × 10⁶	12 weeks
Tran et al., 2020 [30]	Knock-in	RNP	AAV6	ELANE	PB	NOG-XL	Tail vein	0.2 × 10⁶	12–17 weeks
Rai et al, 2020 [31]	Knock-in	RNP	AAV6	ELANE	PB	NSG	Tail vein	0.5 × 10⁶	14 weeks
Rocca et al, 2020 [32]	Knockout	RNP	No donor template	FXN	PB	NSG	Intrahepatic	1 × 10⁶	12 weeks
Goodwin et al, 2020 [33]	Knock-in	RNP	AAV6	FOXP3	UCB	NSGSGM3	Intrahepatic	0.15–1 × 10⁶	8–14 weeks
Weber et al, 2020 [34]	Knockout	RNP	No donor template	HBG1/HBG2	PB	NSG	Intraperitoneal	1 × 10⁶	16 weeks
Brault et al, 2021 [35]	Knock-in	mRNA/gRNA	AAV	MAGT1	PB	NSGS	Intrahepatic	1–1.5 × 106⁶	16 weeks
Sweeney et al, 2021 [36]	Knock-in	RNP	AAV6	CYBB	PB	NSG	Tail vein	1–2 × 10⁶	12 weeks
Uchida et al, 2021 [37]	Knock-in	RNP	ssDNA	HBB	PB	NBSGW	Tail Vein	0.4–0.5 × 10⁶	12–16 weeks
Psatha et al, 2021 [38]	Knockout	RNP	No donor template	BCL11	PB	NBSGW	Unknown	2 × 10⁶	16 weeks
Fananas-Baquero et al, 2021 [39]	Knock-in	RNP	AAV6	PKLR	UCB	NSG	Tail vein	1 × 10⁶	13 weeks
Li et al, 2021 [40]	Knockout	HdAd	No donor template	HBG2	PB	NSG	Tail vein	0.5 × 106⁶	8 weeks
Samuelson et al, 2021 [41]	Knockout	RNP	No donor template	HBG/BCL11	PB	NSG	Tail vein	1 × 10⁶	12–23 weeks
El-Kharrag et al, 2022 [42]	Knockout	RNP	No donor template	CD33	PB	NSG	Tail vein	0.5 × 10⁶	20 weeks
Karrupusamy et al, 2022 [43]	Knockout	RNP	No donor template	CCR5	PB	NBSGW	Tail vein	0.09 × 10⁶	16 weeks
Venkatesan et al,2023 [44]	Knockout	RNP	No donor template	PRR β-globin	PB	NBSGW	Tail vein	0.5–0.6 × 10⁶	16 weeks
Rai et al, 2023 [45]	Knock-in	RNP	AAV6	WAS	PB	NSG	Tail vein	0.5 × 10⁶	8–16 weeks
Wellhausen et al,2023 [46]	Knockout	RNP	No donor template	CD45	PB	NSG	Tail vein	0.5 × 10⁶	12 weeks
Lydeard et al, 2023 [47]	Knockout	RNP	No donor template	CD33	PB	NSG	Tail vein	0.1–1 × 10⁶	16 weeks
Hardouin et al, 2023 [48]	Editing	mRNA/gRNA	No donor template	HBB	PB	NBSGW	Retro-orbital	0.26 × 10⁶	16 weeks
Brault et al, 2023 [49]	Knock-in	mRNA/gRNA	AAV6	IL2RG	PB	NSGS	Intrahepatic	1–1.5 × 10⁶	12–16 weeks
Frati et al, 2024 [50]	Knockout	RNP	No donor template	γ-globin promoter	PB	NSG	Retro-orbital	0.5–2 × 10⁶	16 weeks
Bahal et al, 2024 [51]	Knock-in	RNP	AAV6	BTK	PB	NSG	Tail vein	2 × 10⁶	15 weeks
Demirci et al, 2024 [52]	Knockout	RNP	No donor template	BCL11	PB	NBSGW	Tail vein	0.3 × 10⁶	24 weeks
Nasri et al, 2024 [53]	Knock-in	RNP	ssODN	ELANE	BM	NSG	Intrafemoral	0.1 × 10⁶	16 weeks
Pugliano et al, 2024 [54]	Knock-in	RNP	AAV6	CD40L	PB	NBSGW	Retro-orbital	1.5–2 × 10⁶	12–16 weeks
Dudek et al, 2024 [55]	Knock-in	RNP	AAV6	CCR5	PB	NSGSGM3	Retro-orbital	1 × 10⁶	14 weeks
Park et al, 2019 [56]	Knock-in	Plasmid	ssODN	HBB	PB	NSG	Intrafemoral	0.5 × 10⁶	19 weeks
Dever et al., 2016 [57]	Knock-in	RNP	AAV6	HBB	PB	NSG	Tail vein	0.4–0.7 × 10⁶	Unknown

↓ Table 1. Characteristic Summary of the Preclinical Studies Included in the Metanalysis

Author and year

Goal of gene editing

Cas9 delivery system

Donor template delivery system

Targeted gene

Tissue source of hHSPCs

Mice strain

Mode of transplantation

Number of cells injected

Follow-up duration after transplantation

hHSPCs: human hematopoietic stem and progenitor cells; mRNA: messenger ribonucleic acid; gRNA: guide RNA; RNP: ribonucleoprotein complex; AAV6: adeno-associated virus type 6; ssODN: single-stranded oligodeoxynucleotides; PB: peripheral blood; BM: bone marrow; UCB: umbilical cord blood; CCR5: C-C chemokine receptor type 5; TET2: Tet methylcytosine deoxygenase 2; CYBB: cytochrome b-245 beta chain; HBB: hemoglobin subunit beta; CD: cluster of differentiation; HBG: hemoglobin subunit gamma; ITGB2: integrin beta subunit 2; ELANE: neutrophil elastase; FXN: frataxin; FOXP3: forkhead box P3; MAGT1: magnesium transporter 1; BCL11: B-cell lymphoma/leukemia 11; PKLR: pyruvate kinase L/R; PRR: putative repressor region; WAS: Wiskott Aldrich syndrome; IL2RG: interleukin 2 receptor subunit gamma; BTK: Bruton’s tyrosine kinase; NSG: non-obese diabetic (NOD) Scid gamma.

Xu et al, 2017 [19]

Knockout

Plasmid

No donor template

CCR5

Fetal liver

NSG

Intrahepatic

1 × 10⁶

12 weeks

Tothova et al, 2017 [20]

Knockout

Plasmid

No donor template

TET2/Cohesin

NSGS

Retro-orbital

0.2–0.5 × 10⁶

22 weeks

De Ravin et al, 2017 [21]

Knock-in

mRNA/gRNA

ssODN

CYBB

NSG

Tail vein

1–3 × 10⁶

20 weeks

Yen et al, 2018 [22]

Knockout

RNP

No donor template

HBB

NSG

Tail vein

0.7 × 10⁶

12–20 weeks

Kim et al, 2018 [23]

Knockout

RNP

No donor template

CD33

NSG

Tail vein

0.1–0.5 × 10⁶

16 weeks

Pattabhi et al, 2019 [24]

Knock-in

RNP

AAV6/ssODN

HBB

NBSGW

Tail vein

2 × 10⁶

14 weeks

Borot et al, 2019 [25]

Knockout

RNP

No donor template

CD33

NSGSGM3

Tail vein

0.5–1 × 10⁶

9–21 weeks

Romero et al, 2019 [26]

Knock-in

RNP

AAV6/ssODN

HBB

NSG

Retro-orbital

1–1.3 × 10⁶

16 weeks

Metais et al, 2019 [27]

Knockout

RNP

No donor template

HBG1/HBG2

NBSGW

Tail vein

1 × 10⁶

16–17 weeks

Bloomer et al, 2021 [28]

Knock-in

RNP

AAV6

ITGB2

NSG

Tail vein

0.3 × 10⁶

18 weeks

Yudovich et al, 2020 [29]

Knockout

RNP

No donor template

CD45

UCB

NSG

Tail vein

0.1 × 10⁶

12 weeks

Tran et al., 2020 [30]

Knock-in

RNP

AAV6

ELANE

NOG-XL

Tail vein

0.2 × 10⁶

12–17 weeks

Rai et al, 2020 [31]

Knock-in

RNP

AAV6

ELANE

NSG

Tail vein

0.5 × 10⁶

14 weeks

Rocca et al, 2020 [32]

Knockout

RNP

No donor template

FXN

NSG

Intrahepatic

1 × 10⁶

12 weeks

Goodwin et al, 2020 [33]

Knock-in

RNP

AAV6

FOXP3

UCB

NSGSGM3

Intrahepatic

0.15–1 × 10⁶

8–14 weeks

Weber et al, 2020 [34]

Knockout

RNP

No donor template

HBG1/HBG2

NSG

Intraperitoneal

1 × 10⁶

16 weeks

Brault et al, 2021 [35]

Knock-in

mRNA/gRNA

AAV

MAGT1

NSGS

Intrahepatic

1–1.5 × 106⁶

16 weeks

Sweeney et al, 2021 [36]

Knock-in

RNP

AAV6

CYBB

NSG

Tail vein

1–2 × 10⁶

12 weeks

Uchida et al, 2021 [37]

Knock-in

RNP

ssDNA

HBB

NBSGW

Tail Vein

0.4–0.5 × 10⁶

12–16 weeks

Psatha et al, 2021 [38]

Knockout

RNP

No donor template

BCL11

NBSGW

Unknown

2 × 10⁶

16 weeks

Fananas-Baquero et al, 2021 [39]

Knock-in

RNP

AAV6

PKLR

UCB

NSG

Tail vein

1 × 10⁶

13 weeks

Li et al, 2021 [40]

Knockout

HdAd

No donor template

HBG2

NSG

Tail vein

0.5 × 106⁶

8 weeks

Samuelson et al, 2021 [41]

Knockout

RNP

No donor template

HBG/BCL11

NSG

Tail vein

1 × 10⁶

12–23 weeks

El-Kharrag et al, 2022 [42]

Knockout

RNP

No donor template

CD33

NSG

Tail vein

0.5 × 10⁶

20 weeks

Karrupusamy et al, 2022 [43]

Knockout

RNP

No donor template

CCR5

NBSGW

Tail vein

0.09 × 10⁶

16 weeks

Venkatesan et al,2023 [44]

Knockout

RNP

No donor template

PRR β-globin

NBSGW

Tail vein

0.5–0.6 × 10⁶

16 weeks

Rai et al, 2023 [45]

Knock-in

RNP

AAV6

WAS

NSG

Tail vein

0.5 × 10⁶

8–16 weeks

Wellhausen et al,2023 [46]

Knockout

RNP

No donor template

CD45

NSG

Tail vein

0.5 × 10⁶

12 weeks

Lydeard et al, 2023 [47]

Knockout

RNP

No donor template

CD33

NSG

Tail vein

0.1–1 × 10⁶

16 weeks

Hardouin et al, 2023 [48]

Editing

mRNA/gRNA

No donor template

HBB

NBSGW

Retro-orbital

0.26 × 10⁶

16 weeks

Brault et al, 2023 [49]

Knock-in

mRNA/gRNA

AAV6

IL2RG

NSGS

Intrahepatic

1–1.5 × 10⁶

12–16 weeks

Frati et al, 2024 [50]

Knockout

RNP

No donor template

γ-globin promoter

NSG

Retro-orbital

0.5–2 × 10⁶

16 weeks

Bahal et al, 2024 [51]

Knock-in

RNP

AAV6

BTK

NSG

Tail vein

2 × 10⁶

15 weeks

Demirci et al, 2024 [52]

Knockout

RNP

No donor template

BCL11

NBSGW

Tail vein

0.3 × 10⁶

24 weeks

Nasri et al, 2024 [53]

Knock-in

RNP

ssODN

ELANE

NSG

Intrafemoral

0.1 × 10⁶

16 weeks

Pugliano et al, 2024 [54]

Knock-in

RNP

AAV6

CD40L

NBSGW

Retro-orbital

1.5–2 × 10⁶

12–16 weeks

Dudek et al, 2024 [55]

Knock-in

RNP

AAV6

CCR5

NSGSGM3

Retro-orbital

1 × 10⁶

14 weeks

Park et al, 2019 [56]

Knock-in

Plasmid

ssODN

HBB

NSG

Intrafemoral

0.5 × 10⁶

19 weeks

Dever et al., 2016 [57]

Knock-in

RNP

AAV6

HBB

NSG

Tail vein

0.4–0.7 × 10⁶

Unknown

↓ **Table 2.** Summary of the Pooled Data and Subgroup Analysis of Various Parameters of the Study
Parameter	Groups	Subgroups	Test of heterogeneity	Test model	Types of association	Significance
CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HDR: homology-directed repair; PB: peripheral blood; RNP: ribonucleoprotein complex; BM: bone marrow; UCB: umbilical cord blood; HIV: human immunodeficiency virus; IMD: immune mediated disease; HGP: hemoglobinopathy; SCN: severe congenital neutropenia; AAV6: adeno-associated virus type 6; ssODN: single-strand oligonucleotide; NA: data not available for analysis; HSPC: hematopoietic stem progenitor cell; NS: not significant.
Bone marrow engraftment	Gene editing	Pooled	28.446	1.000	0.000	Fixed	–0.160	–0.292	–0.028	0.018	Significant
		Knock-in	5.681	1.000	0.000	Fixed	0.103	–0.092	0.297	0.302	NS
		Knockout	21.855	0.977	0.000	Fixed	–0.218	–0.401	–0.035	0.020	Significant
	CRISPR-Cas9 system	RNA	0.448	0.998	0.000	Fixed	–0.032	–0.412	0.348	0.869	NS
		RNP	27.505	1.000	0.000	Fixed	–0.177	–0.318	–0.037	0.014	Significant
	Mice strain	NSG	19.312	0.993	0.000	Fixed	–0.238	–0.426	–0.051	0.013	Significant
		NSGS	0.753	0.686	0.000	Fixed	0.324	–0.524	1.172	0.454	NS
		NBSGW	0.885	1.000	0.000	Fixed	–0.055	–0.287	0.176	0.639	NS
		NSGSGM3	4.479	0.812	0.000	Fixed	–0.286	–0.753	0.181	0.229	NS
	Route of transplantation	Tail vein	9.191	1.000	0.000	Fixed	–0.150	–0.314	0.013	0.072	NS
		Intrahepatic	1.290	0.863	0.000	Fixed	–0.011	–0.424	0.402	0.958	NS
		Retro-orbital	14.159	0.166	29.37	Fixed	–0.502	–0.894	–0.109	0.012	Significant
		Intraperitoneal	0.788	0.852	0.000	Fixed	–0.375	–1.078	0.328	0.296	NS
	Source of HSPC	PB	23.709	1.000	0.000	Fixed	–0.166	–0.305	–0.027	0.019	Significant
		BM	3.813	0.577	0.000	Fixed	–0.407	–0.909	0.095	0.112	NS
		UCB	1.634	0.897	0.000	Fixed	0.008	–0.552	0.569	0.976	NS
	Disease targeted	HIV	0.668	0.955	0.000	Fixed	–0.394	–0.987	0.199	0.193	NS
		IMD	3.392	1.000	0.000	Fixed	–0.101	–0.335	0.133	0.397	NS
		HGP	16.408	0.945	0.000	Fixed	–0.199	–0.407	0.009	0.061	NS
		SCN	0.530	0.912	0.000	Fixed	0.092	–0.570	0.755	0.785	NS
		Leukemia	5.806	0.926	0.000	Fixed	–0.183	–0.501	0.134	0.258	NS
	HDR template	AAV6	4.336	1.000	0.000	Fixed	–0.141	–0.350	0.068	0.187	NS
		ssODN	0.387	0.999	0.000	Fixed	0.143	–0.386	0.673	0.596	NS
Spleen engraftment	Gene editing	Pooled	20.908	0.587	0.000	Fixed	–0.307	–0.527	–0.087	0.006	Significant
		Knock-in	7.043	0.532	0.000	Fixed	0.012	–0.269	0.293	0.933	NS
		Knockout	7.881	0.851	0.000	Fixed	–0.663	–1.000	–0.327	< 0.001	Significant
	CRISPR-Cas9 system	RNA	0.997	0.608	0.000	Fixed	0.374	–0.054	0.802	0.087	NS
		RNP	12.694	0.890	0.000	Fixed	–0.457	–0.702	–0.212	< 0.001	Significant
	Mice strain	NSG	5.382	0.864	0.000	Fixed	–0.764	–1.145	–0.383	< 0.001	Significant
		NSGS	NA	NA	NA	NA	NA	NA	NA	NA	NA
		NBSGW	2.518	0.866	0.000	Fixed	–0.147	–0.495	0.200	0.405	NS
		NSGSGM3	0.291	0.962	0.000	Fixed	–0.484	–1.188	0.221	0.178	NS
	Route of transplantation	Tail vein	6.600	0.472	0.000	Fixed	–0.428	–0.837	–0.019	0.040	Significant
		Intrahepatic	NA	NA	NA	NA	NA	NA	NA	NA	NA
		Retro-orbital	3.917	0.917	0.000	Fixed	–0.330	–0.657	–0.004	0.047	Significant
		Intraperitoneal	0.685	0.877	0.000	Fixed	–0.983	–1.721	–0.245	0.009	Significant
	Source of HSPC	PB	NA	NA	NA	NA	NA	NA	NA	NA	NA
		BM	NA	NA	NA	NA	NA	NA	NA	NA	NA
		UCB	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Disease targeted	HIV	0.315	0.989	0.000	Fixed	–0.434	–1.027	0.159	0.152	NS
		IMD	2.139	0.343	6.490	Fixed	0.235	–0.122	0.592	0.196	NS
		HGP	6.797	0.871	0.000	Fixed	–0.479	–0.793	–0.166	0.003	Significant
		SCN	NA	NA	NA	NA	NA	NA	NA	NA	NA
		Leukemia	2.783	0.249	28.140	Fixed	–1.020	–1.919	–0.121	0.026	Significant
	HDR template	AAV6	7.043	0.532	0.000	Fixed	0.012	–0.269	0.293	0.933	NS
		ssODN	NA	NA	NA	NA	NA	NA	NA	NA	NA
Thymus engraftment	Gene editing	Pooled	4.565	0.918	0	Fixed	–0.097	–0.435	0.240	0.572	NS
		Knock-in	3.772	0.287	20.46	Fixed	0.159	–0.174	0.493	0.349	NS
		Knockout	4.009	0.676	0.000	Fixed	–0.087	–0.627	0.452	0.752	NS
	CRISPR-Cas9 system	RNA	3.744	0.287	20.500	Fixed	0.159	–0.174	0.493	0.348	NS
		RNP	4.010	0.675	0.000	Fixed	–0.087	–0.627	0.452	0.751	NS
	Mice strain	NSG	4.288	0.509	0.000	Fixed	–0.153	–0.557	0.251	0.459	NS
		NSGS	1.313	0.859	0.000	Fixed	0.328	–0.070	0.727	0.106	NS
		NBSGW	NA	NA	NA	NA	NA	NA	NA	NA	NA
		NSGSGM3	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Route of transplantation	Tail vein	3.866	0.145	48.260	Fixed	–0.216	–1.075	0.643	0.622	NS
		Intrahepatic	3.847	0.279	22.010	Fixed	0.157	–0.177	0.490	0.357	NS
		Retro-orbital	NA	NA	NA	NA	NA	NA	NA	NA	NA
		Intraperitoneal	0.000	1.000	0.000	Fixed	–0.003	–0.687	0.680	0.993	NS
	Source of HSPC	PB	NA	NA	NA	NA	NA	NA	NA	NA	NA
		BM	NA	NA	NA	NA	NA	NA	NA	NA	NA
		UCB	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Disease targeted	HIV	NA	NA	NA	NA	NA	NA	NA	NA	NA
		IMD	3.773	0.287	20.500	Fixed	0.159	–0.174	0.493	0.349	NS
		HGP	0.040	1.000	0.000	Fixed	0.029	–0.583	0.643	0.924	NS
		SCN	NA	NA	NA	NA	NA	NA	NA	NA	NA
		Leukemia	3.718	0.156	46.210	Fixed	–0.216	–1.074	0.642	0.621	NS
	HDR template	AAV6	3.774	0.287	20.500	Fixed	0.159	–0.174	0.493	0.348	NS
		ssODN	NA	NA	NA	NA	NA	NA	NA	NA	NA
Peripheral blood engraftment	Gene editing	Pooled	12.463	1.000	0.000	Fixed	–0.270	–0.464	–0.075	0.007	Significant
		Knock-in	16.622	0.992	0.000	Fixed	–0.128	–0.307	0.051	0.162	NS
		Knockout	11.536	1.000	0.000	Fixed	–0.231	–0.442	–0.020	0.032	Significant
	CRISPR-Cas9 system	RNA	0.721	0.998	0.000	Fixed	0.348	0.046	0.649	0.024	Significant
		RNP	20.832	1.000	0.000	Fixed	–0.242	–0.388	–0.096	0.001	Significant
	Mice strain	NSG	NA	NA	NA	NA	NA	NA	NA	NA	NA
		NSGS	3.719	0.445	0.000	Fixed	0.313	0.011	0.615	0.042	Significant
		NBSGW	5.341	0.989	0.000	Fixed	–0.233	–0.476	0.010	0.060	NS
		NSGSGM3	0.157	0.984	0.000	Fixed	0.120	–0.481	0.721	0.696	NS
	Route of transplantation	Tail vein	9.513	1.000	0.000	Fixed	–0.207	–0.394	–0.020	0.030	Significant
		Intrahepatic	3.731	0.881	0.000	Fixed	0.224	–0.040	0.488	0.096	NS
		Retro-orbital	5.671	0.579	0.000	Fixed	–0.247	–0.533	0.039	0.090	NS
		Intraperitoneal	0.127	0.988	0.000	Fixed	–0.887	–1.614	–0.160	0.017	Significant
	Source of HSPC	PB	28.135	1.000	0.000	Fixed	–0.172	–0.315	–0.029	0.018	Significant
		BM	0.035	0.983	0.000	Fixed	–0.055	–0.741	0.630	0.875	NS
		UCB	0.157	0.984	0.000	Fixed	0.120	–0.481	0.721	0.696	NS
	Disease targeted	HIV	NA	NA	NA	NA	NA	NA	NA	NA	NA
		IMD	11.636	0.865	0.000	Fixed	–0.128	–0.334	0.078	0.224	NS
		HGP	11.512	1.000	0.000	Fixed	–0.224	–0.432	–0.016	0.035	Significant
		SCN	0.878	0.831	0.000	Fixed	–0.125	–0.789	0.540	0.713	NS
		Leukemia	NA	NA	NA	NA	NA	NA	NA	NA	NA
	HDR template	AAV6	15.384	0.698	0.000	Fixed	–0.082	–0.267	0.103	0.384	NS
		ssODN	3.198	0.994	0.000	Fixed	0.095	–0.359	0.549	0.682	NS

↓ Table 2. Summary of the Pooled Data and Subgroup Analysis of Various Parameters of the Study

Parameter

Groups

Subgroups

Test of heterogeneity

Test model

Types of association

Significance

I² (%)

Hedge’s g

Lower limit

Upper limit

P value

CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HDR: homology-directed repair; PB: peripheral blood; RNP: ribonucleoprotein complex; BM: bone marrow; UCB: umbilical cord blood; HIV: human immunodeficiency virus; IMD: immune mediated disease; HGP: hemoglobinopathy; SCN: severe congenital neutropenia; AAV6: adeno-associated virus type 6; ssODN: single-strand oligonucleotide; NA: data not available for analysis; HSPC: hematopoietic stem progenitor cell; NS: not significant.

Bone marrow engraftment

Gene editing

Pooled

28.446

1.000

0.000

Fixed

–0.160

–0.292

–0.028

0.018

Significant

Knock-in

5.681

1.000

0.000

Fixed

0.103

–0.092

0.297

0.302

Knockout

21.855

0.977

0.000

Fixed

–0.218

–0.401

–0.035

0.020

Significant

CRISPR-Cas9 system

RNA

0.448

0.998

0.000

Fixed

–0.032

–0.412

0.348

0.869

RNP

27.505

1.000

0.000

Fixed

–0.177

–0.318

–0.037

0.014

Significant

Mice strain

NSG

19.312

0.993

0.000

Fixed

–0.238

–0.426

–0.051

0.013

Significant

NSGS

0.753

0.686

0.000

Fixed

0.324

–0.524

1.172

0.454

NBSGW

0.885

1.000

0.000

Fixed

–0.055

–0.287

0.176

0.639

NSGSGM3

4.479

0.812

0.000

Fixed

–0.286

–0.753

0.181

0.229

Route of transplantation

Tail vein

9.191

1.000

0.000

Fixed

–0.150

–0.314

0.013

0.072

Intrahepatic

1.290

0.863

0.000

Fixed

–0.011

–0.424

0.402

0.958

Retro-orbital

14.159

0.166

29.37

Fixed

–0.502

–0.894

–0.109

0.012

Significant

Intraperitoneal

0.788

0.852

0.000

Fixed

–0.375

–1.078

0.328

0.296

Source of HSPC

23.709

1.000

0.000

Fixed

–0.166

–0.305

–0.027

0.019

Significant

3.813

0.577

0.000

Fixed

–0.407

–0.909

0.095

0.112

UCB

1.634

0.897

0.000

Fixed

0.008

–0.552

0.569

0.976

Disease targeted

HIV

0.668

0.955

0.000

Fixed

–0.394

–0.987

0.199

0.193

IMD

3.392

1.000

0.000

Fixed

–0.101

–0.335

0.133

0.397

HGP

16.408

0.945

0.000

Fixed

–0.199

–0.407

0.009

0.061

SCN

0.530

0.912

0.000

Fixed

0.092

–0.570

0.755

0.785

Leukemia

5.806

0.926

0.000

Fixed

–0.183

–0.501

0.134

0.258

HDR template

AAV6

4.336

1.000

0.000

Fixed

–0.141

–0.350

0.068

0.187

ssODN

0.387

0.999

0.000

Fixed

0.143

–0.386

0.673

0.596

Spleen engraftment

Gene editing

Pooled

20.908

0.587

0.000

Fixed

–0.307

–0.527

–0.087

0.006

Significant

Knock-in

7.043

0.532

0.000

Fixed

0.012

–0.269

0.293

0.933

Knockout

7.881

0.851

0.000

Fixed

–0.663

–1.000

–0.327

< 0.001

Significant

CRISPR-Cas9 system

RNA

0.997

0.608

0.000

Fixed

0.374

–0.054

0.802

0.087

RNP

12.694

0.890

0.000

Fixed

–0.457

–0.702

–0.212

< 0.001

Significant

Mice strain

NSG

5.382

0.864

0.000

Fixed

–0.764

–1.145

–0.383

< 0.001

Significant

NSGS

NBSGW

2.518

0.866

0.000

Fixed

–0.147

–0.495

0.200

0.405

NSGSGM3

0.291

0.962

0.000

Fixed

–0.484

–1.188

0.221

0.178

Route of transplantation

Tail vein

6.600

0.472

0.000

Fixed

–0.428

–0.837

–0.019

0.040

Significant

Intrahepatic

Retro-orbital

3.917

0.917

0.000

Fixed

–0.330

–0.657

–0.004

0.047

Significant

Intraperitoneal

0.685

0.877

0.000

Fixed

–0.983

–1.721

–0.245

0.009

Significant

Source of HSPC

UCB

Disease targeted

HIV

0.315

0.989

0.000

Fixed

–0.434

–1.027

0.159

0.152

IMD

2.139

0.343

6.490

Fixed

0.235

–0.122

0.592

0.196

HGP

6.797

0.871

0.000

Fixed

–0.479

–0.793

–0.166

0.003

Significant

SCN

Leukemia

2.783

0.249

28.140

Fixed

–1.020

–1.919

–0.121

0.026

Significant

HDR template

AAV6

7.043

0.532

0.000

Fixed

0.012

–0.269

0.293

0.933

ssODN

Thymus engraftment

Gene editing

Pooled

4.565

0.918

Fixed

–0.097

–0.435

0.240

0.572

Knock-in

3.772

0.287

20.46

Fixed

0.159

–0.174

0.493

0.349

Knockout

4.009

0.676

0.000

Fixed

–0.087

–0.627

0.452

0.752

CRISPR-Cas9 system

RNA

3.744

0.287

20.500

Fixed

0.159

–0.174

0.493

0.348

RNP

4.010

0.675

0.000

Fixed

–0.087

–0.627

0.452

0.751

Mice strain

NSG

4.288

0.509

0.000

Fixed

–0.153

–0.557

0.251

0.459

NSGS

1.313

0.859

0.000

Fixed

0.328

–0.070

0.727

0.106

NBSGW

NSGSGM3

Route of transplantation

Tail vein

3.866

0.145

48.260

Fixed

–0.216

–1.075

0.643

0.622

Intrahepatic

3.847

0.279

22.010

Fixed

0.157

–0.177

0.490

0.357

Retro-orbital

Intraperitoneal

0.000

1.000

0.000

Fixed

–0.003

–0.687

0.680

0.993

Source of HSPC

UCB

Disease targeted

HIV

IMD

3.773

0.287

20.500

Fixed

0.159

–0.174

0.493

0.349

HGP

0.040

1.000

0.000

Fixed

0.029

–0.583

0.643

0.924

SCN

Leukemia

3.718

0.156

46.210

Fixed

–0.216

–1.074

0.642

0.621

HDR template

AAV6

3.774

0.287

20.500

Fixed

0.159

–0.174

0.493

0.348

ssODN

Peripheral blood engraftment

Gene editing

Pooled

12.463

1.000

0.000

Fixed

–0.270

–0.464

–0.075

0.007

Significant

Knock-in

16.622

0.992

0.000

Fixed

–0.128

–0.307

0.051

0.162

Knockout

11.536

1.000

0.000

Fixed

–0.231

–0.442

–0.020

0.032

Significant

CRISPR-Cas9 system

RNA

0.721

0.998

0.000

Fixed

0.348

0.046

0.649

0.024

Significant

RNP

20.832

1.000

0.000

Fixed

–0.242

–0.388

–0.096

0.001

Significant

Mice strain

NSG

NSGS

3.719

0.445

0.000

Fixed

0.313

0.011

0.615

0.042

Significant

NBSGW

5.341

0.989

0.000

Fixed

–0.233

–0.476

0.010

0.060

NSGSGM3

0.157

0.984

0.000

Fixed

0.120

–0.481

0.721

0.696

Route of transplantation

Tail vein

9.513

1.000

0.000

Fixed

–0.207

–0.394

–0.020

0.030

Significant

Intrahepatic

3.731

0.881

0.000

Fixed

0.224

–0.040

0.488

0.096

Retro-orbital

5.671

0.579

0.000

Fixed

–0.247

–0.533

0.039

0.090

Intraperitoneal

0.127

0.988

0.000

Fixed

–0.887

–1.614

–0.160

0.017

Significant

Source of HSPC

28.135

1.000

0.000

Fixed

–0.172

–0.315

–0.029

0.018

Significant

0.035

0.983

0.000

Fixed

–0.055

–0.741

0.630

0.875

UCB

0.157

0.984

0.000

Fixed

0.120

–0.481

0.721

0.696

Disease targeted

HIV

IMD

11.636

0.865

0.000

Fixed

–0.128

–0.334

0.078

0.224

HGP

11.512

1.000

0.000

Fixed

–0.224

–0.432

–0.016

0.035

Significant

SCN

0.878

0.831

0.000

Fixed

–0.125

–0.789

0.540

0.713

Leukemia

HDR template

AAV6

15.384

0.698

0.000

Fixed

–0.082

–0.267

0.103

0.384

ssODN

3.198

0.994

0.000

Fixed

0.095

–0.359

0.549

0.682

↓ **Table 3.** Practical Recommendations for Optimizing Engraftment
	Gene editing	CRISPR-Cas9 system	Mice strain	Route of transplantation	Source of HSPCs	HDR template
CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HSPCs: hematopoietic stem and progenitor cells; HDR: homology-directed repair; RNP: ribonucleoprotein complex; BM: bone marrow; UCB: umbilical cord blood; AAV6: adeno-associated virus type 6; ssODN: single-strand oligonucleotide.
Bone marrow	Knock-in	RNA	NSGS	Tail vein	BM	AAV6
			NBSGW	Intrahepatic	UCB	ssODN
			NSGSGM3	Intraperitoneal
Spleen	Knock-in	RNP	NBSGW	Intrahepatic?	Unknown	AAV6
			NSGSGM3
Thymus	Knock-in	RNA	NSG	Tail vein	Unknown	AAV6
	Knockout	RNP	NSGS	Intrahepatic
				Intraperitoneal
Peripheral blood	Knock-in	Plasmid?	NBSGW	Intrahepatic	BM	AAV6
			NSGSGM3	Retro-orbital	UCB	ssODN

↓ Table 3. Practical Recommendations for Optimizing Engraftment

Gene editing

CRISPR-Cas9 system

Mice strain

Route of transplantation

Source of HSPCs

HDR template

CRISPR-Cas9: clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; HSPCs: hematopoietic stem and progenitor cells; HDR: homology-directed repair; RNP: ribonucleoprotein complex; BM: bone marrow; UCB: umbilical cord blood; AAV6: adeno-associated virus type 6; ssODN: single-strand oligonucleotide.

Bone marrow

Knock-in

RNA

NSGS

Tail vein

AAV6

NBSGW

Intrahepatic

UCB

ssODN

NSGSGM3

Intraperitoneal

Spleen

Knock-in

RNP

NBSGW

Intrahepatic?

Unknown

AAV6

NSGSGM3

Thymus

Knock-in

RNA

NSG

Tail vein

Unknown

AAV6

Knockout

RNP

NSGS

Intrahepatic

Intraperitoneal

Peripheral blood

Knock-in

Plasmid?

NBSGW

Intrahepatic

AAV6

NSGSGM3

Retro-orbital

UCB

ssODN