DEAR EDITOR, Precise gene targeting in monkeys will substantially facilitate generation of human disease models1,2. Although several transgenic monkey models have been successfully

generated3,4,5,6, most of past endeavors in gene targeting using primates had unfortunately failed. The usage of reprogrammable endonucleases, such as ZFN (zinc-finger nuclease), TALEN

(transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system, has opened new avenues for precise gene

modification in monkeys1,2. Among these, the CRISPR/Cas9 system has shown to be most efficacious in gene targeting based on its successful application in a number of animal species7,8.

microinjection of Cas9 mRNA and sgRNAs targeting 3 genes, i.e., _Nr0b1_ (_nuclear receptor subfamily 0 group b member 1_), _Ppar-γ_ (_peroxisome proliferator-activated receptor-γ_), and

_Rag1_ (_recombination activating gene 1_), into one-cell embryos, 10 pregnancies were established from 29 surrogate cynomolgus monkeys (_Macaca fascicularis_). The first set of full-term

delivered twin female infants harbored mutations of _Ppar-γ_ and _Rag1_ but not _Nr0b1_ without any detectable off-target effects9. Monkey genome manipulation using TALEN has been also

reported11. These successes demonstrate the feasibility of precise gene targeting in monkeys. However, to establish genetically modified monkey colony, germline transmission of genetic

modifications is required. While successful germline transmission of transgenes had been reported in monkeys5, it remains to be determined whether CRISPR/Cas9-mediated genome editing in

monkeys can be transmitted to the next generation. As CRISPR/Cas9-mediated genome modifications have been reported to achieve germline transmission in other species, including zebrafish, _C.

elegans_, _Drosophila_, mouse, rat, _etc_.7, germline transmission of Cas9-mediated precise genome modifications in monkeys is highly possible. In our previous study, a pair of twin female

monkeys with _Ppar-γ_ and _Rag1_ mutations were born9. As it takes about 3-4 years for monkeys to reach sexual maturity, we currently cannot perform breeding to assess germline transmission.

In this study, we set out to examine gene targeting in gonads and germ cells to explore whether germline transmission could happen in our Cas9-manipulated monkeys. The total 8 pregnancies

with Cas9-manipulated embryos9 comprised 4 triplets, 1 twin, and 3 singlets (Supplementary information, Table S1). Among them, three live infant founders (Supplementary information, Figure

S1A), including two male (founders I and J) and 1 female monkey (founder N), were successfully delivered from 2 singlet and 1 triplet pregnancies at full term either naturally (I) or through

caesarean section (J and N). One female infant (fetus H) from the third singlet pregnancy died after birth. The rest 4 pregnant monkeys all went through miscarriage in different gestation

stages, including one twin on day 116 (fetuses F and G), three triplets on day 84 (fetuses were not collected), 95 (fetuses C, D and E), and 122 (fetuses K, L and M), respectively. Except

for the triplet miscarried on day 84, all other 8 aborted embryos, including 6 males (fetuses C-F, L and M) and 2 females (fetuses G and K), and the aforementioned dead female infant (fetus

H) were collected (Supplementary information, Table S1). It is worth noting that Cas9 injection led to compatible pregnancy rate but low birth rate12. Considering that the cynomolgus macaque

is a monotocous species, multiple pregnancies may affect pregnancy success. Indeed, of the 8 pregnancies, all 3 singlets were delivered at full term, whereas all multiple pregnancies,

except for one triplet, went through miscarriage (Supplementary information, Table S1). Even though a selective foeticide procedure was applied to 3 triplet pregnancies (recipient No.

070092, 070220 and 080260) to surgically destroy extra embryos to ensure pregnancy success, only one pregnancy went to full term with the birth of a healthy baby, and the other two failed

and were aborted (Supplementary information, Table S1). We previously reported the analyses of limited collectable tissues from the aforementioned 2 live monkeys9. With the aborted embryos,

we were able to determine the integration of Cas9-mediated modifications in different tissues of monkey embryos, and provide direct evidence to support the conclusion that mutations were

wide-spread across different tissues. Given that no _Nr0b1_ mutation was detected in all live monkeys, we focused our analyses on targeted modifications of _Ppar-γ_ and _Rag1_. By PCR

amplification, T7EN1 cleavage assay, and sequencing, we extensively analyzed targeted mutagenesis in 10 somatic tissues, including heart, liver, spleen, kidney, brain, small intestine,

skeletal muscle, placenta, tail and finger, from the two first aborted fetuses C and D. T7EN1 assay showed cleavage bands of _Ppar-γ_ in every tissue of both fetuses C and D except for the

placenta (Figure 1A). Cleavage bands of _Rag1_ were also observed in every tissue from fetus C, indicating that Cas9-mediated mutation could occur in various tissues derived from all three

germ layers. Targeted mutations in different tissues were further confirmed by sequencing analysis (Figure 1C and data not shown), demonstrating the extensive integration of Cas9-mediated

gene editing. Targeted mutagenesis was also observed across various tissues in other aborted embryos (fetuses E-H and K-M; Figure 1B and Supplementary information, Figure S1D-S1G). In sum,

targeted mutagenesis was detected in 8 out of 9 aborted fetuses with efficiency of 7/9 for _Ppar-γ_ (fetuses C, D, E, H, K, L and M), and 4/9 for _Rag1_ (fetuses C, E, G and H). Further

analysis revealed that 3 out of 9 fetuses harbor mutations for both _Ppar-γ_ and _Rag1_ (fetuses C, E and H; Figure 1A, 1B and Supplementary information, Figure S1D and S1E), demonstrating

the potency of the CRISPR/Cas9 method in monkeys. Meanwhile, as described before9, Cas9-mediated targeted mutagenesis of _Ppar-γ_ and _Rag1_ was carefully analyzed in the placenta, umbilical

cord and ear punch tissues of the 3 live infant monkeys. The results indicated that no targeted mutagenesis was detected in founders I and J. The cleavage bands of _Rag1_, but not _Ppar-γ_,

were observed in the placenta of founder N in the T7EN1 cleavage assay (Supplementary information, Figure S1B). Targeted mutations were further confirmed by sequencing of PCR products

(Supplementary information, Figure S1C). These results provide further evidence to confirm the feasibility of using the CRISPR/Cas9 system for monkey gene targeting. As observed before9,

multiple genotypes of _Ppar-γ_ were detected in founder E (Supplementary information, Figure S1D and S1E), further confirming the mosaicism of Cas9-mediated genome targeting in monkeys. The

efficient integration of targeted mutations into different tissues strongly suggests the possibility of gene editing in the gonads. To test this, the testes were isolated from the 2 aborted

male embryos C and D, and the targeted mutagenesis was analyzed by the T7EN1 assay. Consistent with the observations in other somatic tissues, the cleavage of _Rag1_ occurred in the testes

from fetus C, and the cleavage of _Ppar-γ_ was observed in the testes from both fetuses C and D (Figure 1D), demonstrating that Cas9-mediated genome modification was successfully integrated

into gonads of the fetuses. The findings in the males encouraged us to assess the outcome in the females. The ovaries of the aborted female embryos H and K were isolated for analysis of

targeted mutagenesis. The T7EN1 assay showed cleavage bands of _Ppar-γ_ and _Rag1_ in ovaries of fetus H (Figure 1D), which were also observed in various somatic tissues of fetus H (Figure

1B). These data confirm that Cas9-mediated genome modification was integrated into both male and female gonads with efficiency of 3/4 of the tested fetuses. Targeted mutations were further

confirmed by sequencing, which indicated that targeted mutations in the testis, as well as the ovary were the same as those detected in somatic tissues, such as a 1-bp insertion of _Ppar-γ_,

a 6-bp deletion of _Rag1_ in the testes, as well as a 1-bp insertion of _Ppar-γ_, and a 12-bp deletion of _Rag1_ in the ovaries. No mutation was detected in a total of 27 colonies in

_Ppar-γ_ locus of fetus C, which showed weak T7EN1 cleavage bands (Figure 1E and data not shown). Also, sequencing results showed Cas9-mediated gene targeting in the gonads with different

efficiencies. In the testes, the mutagenesis efficiencies were 6.67% for _Ppar-γ_ in fetus D (2/30), and 33.33% for _Rag1_ in fetus C (5/15); in the ovaries, the targeting efficiencies were

36.36% for _Ppar-γ_ (8/22) and 13.04% for _Rag1_ in fetus H (3/23; Figure 1E). The fact that targeted mutations were extensively integrated into gonads strongly suggests a potential that

Cas9-mediated targeting could occur in germ cells. To evaluate this potential, germ cells were isolated from ovaries of aborted embryos H and K, and confirmed by immunostaining with Mvh, a

germ cell specific marker13 (Supplementary information, Figure S1H and S1I). Subsequently, two populations of 7 single germ cells from each aborted fetus were used for detection of

mutations. Consistently, T7EN1 cleavage bands of _Rag1_ were observed in germ cell population from embryo H, and cleavage bands of _Ppar-γ_ were detected in germ cell populations from both

embryos H and K (Figure 1F). The sequencing results further confirmed that these germ cells harbored the same mutations as somatic cells, such as a 1-bp insertion in _Ppar-γ_ (5/24 in fetus

H, 2/14 in fetus K), and a 12-bp deletion in _Rag1_ (16/23 in fetus H; data not shown). These data provide preliminary evidence that Cas9-mediated monkey gene modifications occurred in germ

cells. Remarkably, both tested germ cell populations from 2 different fetuses harbored targeted mutations, demonstrating a high probability of targeted mutations occurring in germ cells.

More supportive evidence was obtained through analysis of individual germ cells. After amplification of the whole genomes from 8 individual germ cells from embryo H, the amplified products

were subjected to mutation analyses as described above. Cleavage bands of _Ppar-γ_ or _Rag1_ were founded in one and two of the 8 reactions of the T7EN1 assay, respectively (Figure 1G),

suggesting that targeted mutations occurred in individual germ cells. Sequencing data further confirmed targeted mutations, such as a 1-bp insertion of _Ppar-γ_, and a 12-bp deletion of

_Rag1_, which are the same as those detected in the somatic cells and germ cell populations of fetus H (Figure 1H). The targeting efficiency is about 50%, suggesting that mutations likely

occur at only one allele in diploid germ cells (Figure 1H). Taken together, targeted mutagenesis detected in the gonad, germ cell population, and single germ cell strongly suggest the

possibility of germline transmission of Cas9-mediated genome modifications in monkeys. In summary, our follow-up studies further confirm the feasibility of using the CRISPR/Cas9 system for

gene editing in monkeys. Furthermore, our data demonstrate that Cas9-mediated mutagenesis extensively occurred in various somatic tissues as well as gonads. Most importantly, our study

provides imperative evidence at both population and single cell levels that Cas9-mediated gene modifications occur in monkey germline successfully, suggesting that it is highly likely that

these modifications could be transmitted through monkey germlines to the next generation. Nevertheless, direct evidence for germline transmission is yet to be obtained. REFERENCES * Chan AW

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Angeles) for careful reading and editing of the manuscript. This study was supported by the National Basic Research Program of China (2011CB944301 and 2012CBA01300), and the National High

Technology Research and Development Program of China (2012AA020701). AUTHOR INFORMATION Author notes * Yongchang Chen, Yiqiang Cui, Bin Shen and Yuyu Niu: These four authors contributed

equally to this work. AUTHORS AND AFFILIATIONS * Yunnan Key Laboratory of Primate Biomedical Research, Kunming, 650500, Yunnan, China Yongchang Chen, Yuyu Niu & Weizhi Ji * Department of

Histology and Embryology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 210029, Jiangsu, China Yiqiang Cui, Bin Shen, Lei Wang, Jianying Wang & 

Jiahao Sha * MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing, 210061, Jiangsu,

China Bin Shen & Xingxu Huang * State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China Xiaoyang Zhao, Wei Li & Qi

Zhou * Kunming Biomed International and National Engineering Research Center of Biomedicine and Animal Science, Kunming, 650500, Yunnan, China Yongchang Chen, Yuyu Niu & Weizhi Ji * Life

Science and Technology, Kunming University of Science and Technology, Kunming, 650224, Yunnan, China Yongchang Chen, Yuyu Niu & Weizhi Ji * Shanghai Key Laboratory of Reproductive

Medicine, Shanghai, 200025, China Xingxu Huang Authors * Yongchang Chen View author publications You can also search for this author inPubMed Google Scholar * Yiqiang Cui View author

publications You can also search for this author inPubMed Google Scholar * Bin Shen View author publications You can also search for this author inPubMed Google Scholar * Yuyu Niu View

author publications You can also search for this author inPubMed Google Scholar * Xiaoyang Zhao View author publications You can also search for this author inPubMed Google Scholar * Lei

Wang View author publications You can also search for this author inPubMed Google Scholar * Jianying Wang View author publications You can also search for this author inPubMed Google Scholar

* Wei Li View author publications You can also search for this author inPubMed Google Scholar * Qi Zhou View author publications You can also search for this author inPubMed Google Scholar

* Weizhi Ji View author publications You can also search for this author inPubMed Google Scholar * Jiahao Sha View author publications You can also search for this author inPubMed Google

Scholar * Xingxu Huang View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Weizhi Ji, Jiahao Sha or Xingxu Huang.

ADDITIONAL INFORMATION ( SUPPLEMENTARY INFORMATION is linked to the online version of the paper on the _Cell Research_ website.) SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION, FIGURE

S1 (A) Photographs of 69-day-old founder I, 64-day-old founder J and 31-day-old founder N. (PDF 618 kb) SUPPLEMENTARY INFORMATION, TABLE S1 Summary of live founders and aborted fetuses (PDF

102 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chen, Y., Cui, Y., Shen, B. _et al._ Germline acquisition of Cas9/RNA-mediated gene modifications

in monkeys. _Cell Res_ 25, 262–265 (2015). https://doi.org/10.1038/cr.2014.167 Download citation * Published: 23 December 2014 * Issue Date: February 2015 * DOI:

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