Generation of Cynomolgus and Rhesus Monkey Models Carrying the BMPR1BFecB Variant

doi:10.12300/j.issn.1674-5817.2026.122

Abstract

Abstract:

Objective To generate cynomolgus and rhesus monkey models carrying the Booroola fecundity (FecB) variant of bone morphogenetic protein receptor type 1B (BMPR1B) gene (designated BMPR1B^FecB ) using adenine base edit technology and to explore genetic improvement strategies for enhancing the reproductive performance of laboratory macaques. Methods Four single-guide RNAs (sgRNAs) were designed to target the BMPR1B c.746A>G locus in cynomolgus and rhesus monkeys. The sgRNAs were combined with the ABE8e-SpRY base editor, and the resulting editing complexes were microinjected into zygotes of both macaque species. Following in vitro culture of the injected embryos, nested PCR amplification was performed to detect the base-editing efficiency at the target site to screen for highly efficient sgRNAs. Subsequently, normally developing gene-edited embryos were transferred into the oviducts of surrogates. Seven days after birth, ear skin or peripheral blood samples were collected from the newborn monkeys for genotyping and mosaicism analysis, and the growth, development, and health status of the gene-edited monkeys were monitored. Results Among the four candidate sgRNAs, sgRNA4 was identified as achieving 100% editing efficiency at the c.746A>G site. A total of 17 live monkey offspring were obtained via embryo transfer, including 8 cynomolgus monkeys and 9 rhesus monkeys. Genotyping results showed that all 8 newborn cynomolgus monkeys carried the c.746A>G point mutation, with an editing efficiency of 100% (8/8), of which 37.50% (3/8) were mosaics. Among the 9 newborn rhesus monkeys, 8 individuals carried the c.746A>G mutation, with an editing efficiency of 88.89% (8/9), of which 75.00% (6/8) were mosaics. Conclusion The ABE8e-SpRY base-editing system enables precise editing at the c.746A>G (p.Q249R) locus of the BMPR1B gene in both cynomolgus and rhesus monkey embryos, resulting in the successful establishment of laboratory monkey models carrying the BMPR1B^FecB variant. These models provide a valuable foundation for future studies of the regulatory role of the BMPR1B^FecB variant in reproductive performance at the non‑human primate level, as well as for exploring genetic improvement strategies to enhance reproductive performance in laboratory monkeys.

Key words: Bone morphogenetic protein receptor type 1B, Base editing, BMPR1B^FecB variant, Cynomolgus monkey, Rhesus monkey

CLC Number:

Q95-33

LI Chunyang,LIU Xingchen,LI Yuzhuo,et al. Generation of Cynomolgus and Rhesus Monkey Models Carrying the BMPR1B^FecB Variant[J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 467-475. DOI: 10.12300/j.issn.1674-5817.2026.122.

Figures/Tables 6

Figure 1 Design and screening of sgRNAs targeting the c.746A>G locus in BMPR1B gene
Note： A， Design of target-site sgRNAs； B， Editing results of the 4 sgRNAs， and the target loci are indicated within red boxes. BMPR1B， bone morphogenetic protein receptor type 1B； WT， wild type； Q249R， c.746A>G mutation； sgRNA， single-guide RNA.

Table 1 Base-editing efficiencies of different sgRNAs in monkey embryos obtained by microinjection

sgRNA编号 sgRNA ID	编辑物种 Edited species	靶位点编辑效率/% Target-site editing efficiency/%	纯合编辑效率/% Homozygous editing efficiency/%	旁位编辑 Bystander editing
sgRNA1	MFA	28.57	0.00	Yes
sgRNA2	MFA	70.00	0.00	No
sgRNA3	MFA	0.00	0.00	Yes
sgRNA4	MFA	100.00	55.56	No
sgRNA4	MMU	100.00	91.67	No

Table 2 Embryo transfer and pregnancy outcomes for BMPR1B^FecB gene-edited embryos

物种

Species

移植胚胎数/枚

Transferred embryos

移植受体数/只

Surrogates

妊娠母体

数/只

Pregnancies

妊娠率/%

Pregnancy rate/%

胎儿数/只

Fetuses

活产数/只

Live births

活产率/%

Live birth rate/%

存活数/只

Surviving offspring

编辑数/只

Edited offspring

编辑效率/%

Editing efficiency/%

Figure 2 Representative genotypes of newborn BMPR1B^FecB gene-edited monkeys
Note： A， Genotypes of ear-tip tissue and blood samples from cynomolgus monkey #0673； B， Genotypes of ear-tip tissue and blood samples from rhesus monkey #H0806. Target loci are highlighted in red boxes. MFA， Macaca fascicularis；MMU， Macaca mulatta；WT， wild type.

Table 3 Mosaic ratios in surviving BMPR1B^FecB gene-edited monkeys

动物编号 Animal ID	性别 Sex	基因型 Genotype	个体嵌合率/% Mosaicism rate/%
0673	Male	Hom	100.00
0677	Male	Hom	100.00
0684	Male	Hom	100.00
0687	Female	Hom	100.00
0689	Female	Mos	75.36
0693	Male	Mos	97.18
0782	Female	Mos	92.50
0783	Female	Hom	100.00
H0507	Male	Mos	62.77
H0509	Male	Mos	52.34
H0611	Male	Mos	88.51
H0813	Male	WT	0.00
H0815	Male	Mos	48.65
H0806	Female	Hom	100.00
H0819	Male	Hom	100.00
H0823	Male	Mos	67.95
H0911	Male	Mos	41.59

Figure 3 Healthy surviving BMPR1B^FecB gene-edited monkeys
Note： A， BMPR1B^FecB gene-edited cynomolgus monkeys （females，3-month-old， twins）； B， BMPR1B^FecB gene-edited rhesus monkey （female， 3-month-old）.

References 14

[1]	孙强. 中国实验猴产业的历史、现状、挑战与机遇[J]. 实验动物与比较医学, 2024, 44(4): 343-356. DOI: 10.12300/j.issn.1674-5817.2024.112 .
	SUN Q. History, current status, challenges and opportunities of laboratory monkey industry in China[J]. Lab Anim Comp Med, 2024, 44(4): 343-356. DOI: 10.12300/j.issn.1674-5817.2024.112 .
[2]	National Center for Biotechnology Information. BMPR1B bone morphogenetic protein receptor type 1B [Homo sapiens (human)][DB/OL]. [2026-07-06]. .
[3]	SHIMASAKI S, MOORE R K, ERICKSON G F, et al. The role of bone morphogenetic proteins in ovarian function[J]. Reprod Suppl, 2003, 61: 323-337. DOI: 10.1530/biosciprocs.5.024 .
[4]	EDSON M A, NALAM R L, CLEMENTI C, et al. Granulosa cell-expressed BMPR1A and BMPR1B have unique functions in regulating fertility but act redundantly to suppress ovarian tumor development[J]. Mol Endocrinol, 2010, 24(6): 1251-1266. DOI: 10.1210/me.2009-0461 .
[5]	WILSON T, WU X Y, JUENGEL J L, et al. Highly prolific booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells[J]. Biol Reprod, 2001, 64(4): 1225-1235. DOI: 10.1095/biolreprod64. 4.1225 .
[6]	MULSANT P, LECERF F, FABRE S, et al. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[J]. Proc Natl Acad Sci U S A, 2001, 98(9): 5104-5109. DOI: 10.1073/pnas.091577598 .
[7]	DAVIS G H. Major genes affecting ovulation rate in sheep[J]. Genet Sel Evol, 2005, 37(1): S11-S23. DOI: 10.1186/1297-9686-37-S1-S11 .
[8]	Online Mendelian Inheritance in Animals. Fecundity, BMPR1B-related in Ovis aries (sheep)[DB/OL]. .
[9]	WANG X Y, GUO X F, HE X Y, et al. Effects of FecB mutation on estrus, ovulation, and endocrine characteristics in small tail Han sheep[J]. Front Vet Sci, 2021, 8: 709737. DOI: 10.3389/fvets.2021.709737 .
[10]	ZHOU S W, DING Y G, LIU J, et al. Highly efficient generation of sheep with a defined FecB^B mutation via adenine base editing[J]. Genet Sel Evol, 2020, 52(1): 35. DOI: 10.1186/s12711-020-00554-6 .
[11]	ZHANG X M, QIU M Y, HAN B, et al. Generation and propagation of high fecundity gene edited fine wool sheep by CRISPR/Cas9[J]. Sci Rep, 2025, 15: 2557. DOI: 10.1038/s41598-025-86592-w .
[12]	SUN Q, DONG J, YANG W T, et al. Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique[J]. Proc Natl Acad Sci U S A, 2008, 105(35): 12956-12960. DOI: 10.1073/pnas.0805639105 .
[13]	HUANG T P, HEINS Z J, MILLER S M, et al. High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs[J]. Nat Biotechnol, 2023, 41(1): 96-107. DOI: 10.1038/s41587-022-01410-2 .
[14]	LU Z Y, HE S T, JIANG J, et al. Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy[J]. Mol Ther, 2022, 30(6): 2163-2175. DOI: 10.1016/j.ymthe. 2022.03.001 .

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