Background: Polycystic ovary syndrome (PCOS) is a frequently encountered endocrine disturbance with a still poorly defined etiology that arises in women during their reproductive years. Increased apoptosis of granulosa cells has been identified as one of the key factors contributing to abnormal follicular development. This study aimed to elucidate the role of six-transmembrane epithelial antigen of prostate 4 (STEAP4) in granulosa cell function using in vitro and in vivo models relevant to PCOS. Methods: We treated KGN cells (a human granulosa-like cell line) and C57BL/6 mice with dehydroepiandrosterone (DHEA) to establish experimental models mimicking PCOS features. STEAP4 expression was assessed by qRT-PCR, Western blot, and immunohistochemistry. Proliferative capacity and apoptotic rates were gauged with CCK-8 assays, EdU labeling, and flow cytometry. The regulatory mechanisms were investigated through immunofluorescence staining for nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and immunoprecipitation assays for HIF-1α ubiquitination. Results: Exposure to androgen markedly raised both STEAP4 transcript and protein abundance in KGN cells as well as in PCOS model mice. STEAP4 knockdown resulted in increased proliferation and reduced apoptosis in DHEA-treated KGN cells. Mechanistically, STEAP4 enhanced reactive oxygen species levels, promoted Nrf2 nuclear translocation, and stabilized HIF-1α protein by reducing its ubiquitination, leading to increased TERT expression and subsequent TP53 pathway activation. In vivo, STEAP4 silencing significantly alleviated hormonal imbalances, estrous cycle disorders, and reduced oxidative stress levels in ovarian tissue of DHEA-induced PCOS-like mice. Conclusions: Taken together, evidence from these experimental models indicates that STEAP4 shapes oxidative stress and granulosa cell apoptosis by operating through the ROS-TERT-TP53 axis. The data point to a possible contribution of STEAP4 to PCOS pathogenesis and mark it as a candidate therapeutic target that merits additional clinical study.

Telomere length (TL) is important for maintaining the individual health of a species. Recent studies shows that in vitro fertilization therapy can drastically reduce TL in offspring, however, the underlying molecular mechanism remains unknown. Sirt6 is a NAD+-dependent epigenetic regulator that has recently been found to play an important role in maintaining telomere stability. Here, we report that NAD+ deficiency in in vitro-cultured blastocysts impairs Sirt6 function, triggering telomere shortening of the inner cell mass and possibly affecting newborns. This phenotype could be effectively mitigated by supplementation with nicotinamide mononucleotide (NAD+ precursor) during in vitro culture, while it could not be achieved in Sirt6 conditional knockout embryos. mtROS accumulation and epigenetic modifications may also be involved in this process. Our results reveal the mechanism by which in vitro culture induces telomere shortening in preimplantation embryos, providing a potential target for improving in vitro culture conditions.

Mutations in mitochondrial DNA (mtDNA) can lead to mitochondrial and cellular dysfunction. However, recent studies suggest that purifying selection acts against mutant mtDNAs during transgenerational transmission. We investigated the mtDNA dynamics during ovarian follicle development. Using base-editing, we generated mice harboring a 3177 G > A mutation corresponding to the human Leber hereditary optic neuropathy (LHON)-related mtDNA mutation and confirmed a transgenerational reduction of the mutant mtDNA. Utilizing a mouse follicle culture system in which pathogenic mtDNA mutations were introduced in vitro, followed by mtDNA sequencing and digital PCR, we found that the germline heteroplasmy shift during early folliculogenesis was driven by a decrease in mutant mtDNA along with compensatory replication of wild-type mtDNA. In contrast, synonymous mtDNA mutations did not affect mtDNA dynamics. These findings demonstrate that mice can eliminate certain pathogenic mtDNA mutations in the germline during early folliculogenesis, thus advancing our understanding of mtDNA purifying selection during oogenesis. Furthermore, our use of mtDNA editing in in vitro-cultured follicles provides a novel approach to create and monitor mitochondrial DNA mutations.

RESEARCH QUESTION:Is cell-free mitochondrial DNA (cf-mtDNA) in spent blastocyst medium (SBM) a suitable biomarker for non-invasive preimplantation genetic testing (niPGT) for mtDNA diseases, and what factors influence the accuracy of niPGT results? DESIGN:Oocytes isolated from mouse models were fertilized and cultured to the blastocyst stage using a two-step sequential culture procedure. For each blastocyst, SBM and the corresponding embryo were collected, and trophectoderm biopsies were performed exclusively on high-quality blastocysts. These samples were subsequently analysed using real-time polymerase chain reaction, or subjected to mtDNA direct amplification for next-generation sequencing. RESULTS:The amplification rate of the target mtDNA site in SBM was 100% in both mice and humans, whereas the amplification rate of the GAPDH gene within the human nuclear genome was 75%. Linear regression analysis revealed strong correlation of the trophectoderm biopsy group (r² = 0.96, prediction interval 6.87 ± 0.06%) and the cf-mtDNA group (r² = 0.84, prediction interval 14.29 ± 0.11%) with the remaining whole embryo. However, in SBM from low-quality embryos, the r2-value was lower (r² = 0.17, prediction interval 37.80 ± 0.60%) compared with SBM from high-quality embryos (r² = 0.84, prediction interval 14.29 ± 0.11%). Notably, the sequential culture procedure minimized contamination from cumulus cells, and SBM samples contaminated with fewer than 10 spermatozoa did not affect the niPGT results. CONCLUSION:Based on the sequential culture procedure, niPGT may serve as an alternative method for preventing maternal-to-offspring transmission of mtDNA diseases. However, this conclusion was based on an animal model, and the findings require further validation in humans.

Introduction: Mitochondrial diseases caused by mtDNA have no effective cures. Recently developed DddA-derived cytosine base editors (DdCBEs) have potential therapeutic implications in rescuing the mtDNA mutations. However, the performance of DdCBEs relies on designing different targets or improving combinations of split-DddA halves and orientations, lacking knowledge of predicting the results before its application.Methods: A series of DdCBE pairs for wide ranges of aC or tC targets was constructed, and transfected into Neuro-2a cells. The mutation rate of targets was compared to figure out the potential editing rules.Results: It is found that DdCBEs mediated mtDNA editing is predictable: 1) aC targets have a concentrated editing window for mtDNA editing in comparison with tC targets, which at 5’C8-11 (G1333) and 5’C10-13 (G1397) for aC target, while 5’C4-13 (G1333) and 5’C5-14 (G1397) for tC target with 16bp spacer. 2) G1333 mediated C>T conversion at aC targets in DddA-half-specific manner, while G1333 and G1397 mediated C>T conversion are DddA-half-prefer separately for tC and aC targets. 3) The nucleotide adjacent to the 3’ end of aC motif affects mtDNA editing. Finally, by the guidance of these rules, a cell model harboring a pathogenic mtDNA mutation was constructed with high efficiency and no bystander effects.Discussion: In summary, this discovery helps us conceive the optimal strategy for accurate mtDNA editing, avoiding time- and effort-consuming optimized screening jobs.

Efficient germline mtDNA editing is required to construct disease-related animal models and future gene therapy. Recently, the DddA-derived cytosine base editors (DdCBEs) have made mitochondrial genome (mtDNA) precise editing possible. However, there still exist challenges for editing some mtDNA sites in germline via zygote injection, probably due to the suspended mtDNA replication during preimplantation development. Here, we introduce a germline mtDNA base editing strategy: injecting DdCBEs into oocytes of secondary follicles, at which stage mtDNA replicates actively. With this method, we successfully observed efficient G-to-A conversion at a hard-to-edit site and also obtained live animal models. In addition, for those editable sites, this strategy can greatly improve the base editing efficiency up to 3-fold, which is more than that in zygotes. More important, editing in secondary follicles did not increase more the risk of off-target effects than that in zygotes. This strategy provides an option to efficiently manipulate mtDNA sites in germline, especially for hard-to-edit sites.

Background Techniques for sperm cryopreservation have exhibited their potential in male fertility preservation. The use of frozen-thawed sperm in in vitro fertilization (IVF) cycles is widespread today. However, many studies reported that cryopreservation might have adverse effects on sperm DNA integrity, motility, and fertilization, probably due to cold shock, intra- and extracellular ice crystals, and excess reactive oxygen species (ROS). Studies suggested that freezing and thawing impaired sperm viability and might adversely affect subsequent fertilization and pregnancy outcomes. The potential damage to fertilization and subsequent embryonic development and offspring health raises the concern on sperm cryopreservation. However, the above mentioned studies are limited to intracytoplasmic sperm injection (ICSI) cycles, while IVF is a more natural and patient-friendly method. IVF requires a higher quality of sperm than ICSI. However, the effect of freezing and thawing on sperm used for IVF remains unknown. Therefore, we aim to investigate the effect of freezing and thawing on ejaculated sperm and subsequent pregnancy and neonatal outcomes in IVF. Methods This retrospective cohort study at a tertiary-care academic medical center included 447 women who used paternal frozen-thawed ejaculated sperm and 31,039 women who used paternal freshly ejaculated sperm for IVF and underwent frozen-thawed blastocyst transfer from January 2011 to September 2021. To balance the baseline characteristics of the two groups, patients using frozen sperm were matched with control groups using a propensity score matching algorithm with a ratio of 1:3. Results Although sperm motility decreased from 82.04% to 75.70% (P < 0.001) after the freezing-thawing process, the fertilization rate (68.27% for frozen sperm and 67.54% for fresh sperm), number of viable embryos (1.98 and 2.16), clinical pregnancy rate (44.7% and 51.8%), and live birth rate (40.3% and 42.4%) were comparable between the two groups (all P > 0.05). For neonatal outcomes, no between-group differences were observed in offspring gender, gestational age, birthweight, and the rate of preterm birth (21.7% and 12.9%), low birthweight neonates (19.2% and 16.0%), and birth defects (0.0% and 0.8%) (all P>0.05). Conclusions Frozen-thawed sperm had lower sperm motility but resulted in comparable embryonic, pregnancy, and neonatal outcomes versus fresh sperm in IVF cycles.

旨在通过构建体外细胞模型研究在长效CRISPR系统作用下基因编辑的动态结局,为在体生殖系长效编辑提供研究支持.本研究以FANCF和VEGFA基因为例,通过慢病毒随机整合的方式构建了一套长效表达CRISPR/Cas9的细胞模型,并以该模型为对象研究长效CRISPR系统作用下,基因编辑效率和修复的动态结局.在FANCF和VEGFA基因外源靶点区域,随着Doxycycline诱导时间的增加,CRISPR/Cas9的编辑效率逐渐升高,并分别于第4天(FANCF)、第3天(VEGFA)编辑效率达到峰值;其次,对应上述靶区的内源靶点的达到峰值的时间点与外源靶点基本一致,但内源靶点的编辑效率显著高于外源靶点(P<0.000 1);再次,对靶点编辑产物序列分析发现,在编辑的第4～7天,各靶点编辑产物的删除、插入及其它类型呈稳定状态,内、外源各位点编辑结局中删除、插入以及其它类型的占比趋势一致;最后,对FANCF靶点潜在的6个脱靶位点进行检测,均未发现任何脱靶现象.长效CRISPR/Cas9技术的编辑效率在第3或第4天达到峰值,各基因编辑结局类型占比趋势一致,且在FANCF位点上不会增加脱靶风险.

Clustered protocadherins (Pcdhs) are a large family of cadherin-like cell adhesion proteins that are central for neurite self-avoidance and neuronal connectivity in the brain. Their downstream nonreceptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2, also known as Ptk2b, Cakb, Raftk, Fak2, and Cadtk) is predominantly expressed in the hippocampus. We constructed Pyk2-null mouse lines and found that these mutant mice showed enhancement in contextual fear memory, without significant change in auditory-cued and spatial-referenced learning and memory. In addition, by preparing Y402F mutant mice, we observed that Pyk2 suppressed contextual fear memory in an autophosphorylation-independent manner. Moreover, using high-throughput RNA sequencing, we found that immediate early genes, such as Npas4, cFos, Zif268/Egr1, Arc, and Nr4a1, were enhanced in Pyk2-null mice. We further showed that Pyk2 disruption affected pyramidal neuronal complexity and spine dynamics. Thus, we demonstrated that Pyk2 is a novel fear memory suppressor molecule and Pyk2-null mice provide a model for understanding fear-related disorders. These findings have interesting implications regarding dysregulation of the Pcdh‒Pyk2 axis in neuropsychiatric disorders.

AIMS:Folliculogenesis contains gonadotropin-independent and -dependent stage. Disruption in any of this process would induce failure in retrieving capable oocytes during clinical treatment. However, there is still limited understanding of the molecular components specifically regulating this process.MATERIAL AND METHODS:Ovaries of P3, P20 and exogenous gonadotropin-treated P22 mice were sampled and underwent RNA-seq to investigate the transcriptome variance during mouse folliculogenesis.KEY FINDINGS:In our dataset, 1883 and 626 DEGs were captured for each stage respectively, which were further clustered into eight expression patterns. Pathway enrichment analysis identified distinct biological processes enriched in two stages, with the most prominent being the pathways related to metabolism, gene expression, cell cycle, immune system and DNA methylation. Transcriptional regulator inference yielded eight master transcription factors (i.e. Runx1, Stat3, Sox3, Pou5f1, Gata4, Foxl2, Cebpb, and Esr1) driving folliculogenesis.SIGNIFICANCE:Our study revealed the temporal transcriptional reprogramming and gene expression dynamics during folliculogenesis mediated by extra hormone treatment, which could provide novel insights to controlled ovarian stimulation in future infertility treatment.

STUDY QUESTIONDoes trophectoderm (TE) quality affect birthweight after single frozen-thawed blastocyst transfer?SUMMARY ANSWERTransfer of single blastocyst with advanced TE quality was associated with higher birthweight and increased risk of a large for gestational age (LGA) baby.WHAT IS KNOWN ALREADYTransfer of blastocysts with advanced TE quality results in higher ongoing pregnancy rates and a lower miscarriage risk. However, data on the relationship between TE quality and birthweight are still lacking.STUDY DESIGN, SIZE, DURATIONThis retrospective cohort study at a tertiary-care academic medical center included 1548 singleton babies born from single frozen-thawed blastocyst transfer from January 2011 to June 2019.PARTICIPANTS/MATERIALS, SETTING, METHODSBabies were grouped into four groups according to embryo expansion (Stages 3, 4, 5 and 6), three groups according to inner cell mass (ICM) quality (A, B and C), and three groups according to TE quality (A, B and C). Main outcomes included absolute birthweight, Z-scores adjusted for gestational age and gender, and adverse neonatal outcomes. Multivariable linear and logistic regression analyses were performed to investigate the association of neonatal outcomes with expansion stage, ICM quality and TE quality.MAIN RESULTS AND THE ROLE OF CHANCEAs TE quality decreased, birthweight (3468.10 ± 471.52, 3357.69 ± 522.06, and 3288.79 ± 501.90 for A, B and C, respectively, P = 0.002), Z-scores (0.59 ± 1.07, 0.42 ± 1.04, and 0.27 ± 1.06 for A, B and C, respectively, P = 0.002) and incidence of LGA (28.9%, 19.7% and 17.4% for A, B and C, respectively, P = 0.027) decreased correspondingly. After adjusting for confounders, compared with the Grade A group, blastocysts with TE Grade B (standardized coefficients (β): -127.97 g, 95% CI: -234.46 to -21.47, P = 0.019) and blastocysts with TE grade C (β: -200.27 g, 95% CI: -320.69 to -79.86, P = 0.001) resulted in offspring with lower birthweight. Blastocysts with TE grade C brought babies with lower Z-scores than TE Grade A (β: -0.35, 95% CI: -0.59 to -0.10, P = 0.005). Also, embryos with TE Grade B (adjusted odds ratio (aOR):0.91, 95% CI: 0.84 to 0.99, P = 0.033) and embryos with TE Grade C (aOR : 0.89, 95% CI: 0.81 to 0.98, P = 0.016) had lower chance of leading to a LGA baby than those with TE Grade A. No association between neonatal outcomes with embryo expansion stage and ICM was observed (all P > 0.05).LIMITATIONS, REASONS FOR CAUTIONThe retrospective design, lack of controlling for several unknown confounders, and inter-observer variation limited this study.WIDER IMPLICATIONS OF THE FINDINGSThe study extends our knowledge of the down-stream effect of TE quality on newborn birthweight and the risk of LGA.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by National Key R&D Program of China (2018YFC1003000), National Natural Science Foundation of China (81771533 to Y.P.K. and 31200825 to L.S.) and Innovative Research Team of High-level Local Universities in Shanghai (SSMU-ZLCX20180401), Shanghai Sailing Program(21YF1423200) and the Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (JYZZ117). The authors declare no conflict of interest in this present study.TRIAL REGISTRATION NUMBERN/A.

Introduction:As an effective inhibitor of premature ovulation, progestin was introduced to a novel ovarian stimulation regimen for infertility treatment. However, the local action of progestin on the ovary and its effect on clinical outcomes have not been described.Objectives:The influence of progesterone administration on clinical oocyte outcomes and the mechanisms involved in the coordination of progesterone and follicle stimulating hormone (FSH) on follicle growth and oocyte yields were investigated.Methods:Clinical outcomes of patients undergoing ovarian stimulation for in vitro fertilization were analyzed. The murine ovarian stimulation model and follicle culture system were used to evaluate the effects of progesterone on oocyte yield, follicle development, granular cell proliferation, and hormone secretion. Phospho-specific protein microarrays were used to explore involved signaling pathways.Results:Progesterone decreased clinical oocyte yields, and yields were rescued with an increased dose of human menopausal gonadotropin. Administration of progesterone inhibited murine granular cell proliferation and reduced the growth rate of follicles; both of which were rescued by FSH. The phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) were identified as pivotal signaling pathways to integrate progesterone into the FSH signaling network in granular cells.Conclusion:Progesterone inhibited granular cell proliferation and antral follicle growth during ovarian stimulation, and subsequently influenced oocyte outcomes in the clinical setting. Progesterone coordinated with FSH to regulate follicle growth through PI3K/AKT and MAPK signaling pathways. These findings advance our knowledge regarding the ovarian response to gonadotropins during progestin-primed ovarian stimulation and create an opportunity to manipulate individual oocyte yields.

Ptk2b has been found playing critical roles in oocyte maturation and subsequent fertilization in vitro. But what is the exact in vivo function in reproduction still elusive. Here, by constructing Ptk2b mutant mice, we found Ptk2b was not essential for mice fertility, unexpectedly, contrary to previously reported in vitro findings, we found Ptk2b ablation significantly improved female fecundity. Follicle counting indicated that the number of primordial follicles and growing follicles in matured mice was significantly increased in the absence of Ptk2b, whereas the primordial follicle formation showed no defects. We also found this regulation was in an autophosphorylation independent pathway, as autophosphorylation site mutant mice (PTK2BY402F ) show no phenotype in female fertility. Further biochemistry studies revealed that Ptk2b ablation promotes folliculogenesis via Erk pathway mediate follicle survival. Together, we found a novel biological function of Ptk2b in folliculogenesis, which could be potentially used as a therapeutic target for corresponding infertility.

We read with great interest the recent study in RBMO (Bakkensen et al., 2020Bakkensen J.B. Christou G. Dimitriadis I. James K. Souter I. The effect of follicular phase length on pregnancy outcomes and endometrial development in gonadotropin ovarian stimulation/intrauterine insemination cycles.Reproductive BioMedicine Online. 2020; 40: 362-368Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar), reporting a positive relationship between follicular phase length and clinical pregnancy for intrauterine insemination using gonadotropin. Although this study is indeed informative, we noticed some issues that should be further addressed. First, the authors state 'the odds of clinical pregnancy increased by 6.0% with each additional day of the follicular phase'. However, as the phase length in this study ranged from 6 to 38 days, is an excessive long follicular phase really better than a shorter one (i.e. 38 versus 12 days)? Excessive long follicular phase (>20 days) has been reported to be associated with luteal deficiency and increased risk of miscarriage (Rodriguez-Rigau et al., 1983Rodriguez-Rigau L.J. Shenoi P.N. Smith K.D. Steinberger E. The relationship between the lengths of the follicular and luteal phases of the menstrual cycle and the fertility potential of the female.Fertility and sterility. 1983; 39: 856-857Abstract Full Text PDF PubMed Google Scholar). Considering the possibly small proportion of patients of this kind, the effect of excessive long follicular phase could be neglected in the regression model. It would be advisable for the authors to perform a fractional polynomial regression for continuous variables, or divide cycles into at least three groups (15th percentile, 75th percentile) for categorical variables. Second, we question why only age, BMI and day 3 FSH were adjusted as confounders. The infertility diagnosis and gonadotropin dose were unbalanced between the two groups and many other factors associated with pregnancy outcomes were not introduced into the final model (Thijssen et al., 2017Thijssen A. Creemers A. Van der Elst W. Creemers E. Vandormael E. Dhont N. Ombelet W. Predictive value of different covariates influencing pregnancy rate following intrauterine insemination with homologous semen: a prospective cohort study.Reproductive biomedicine online. 2017; 34: 463-472Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The length of follicular phase is a comprehensive reflection of patient characteristics (Jukic et al., 2007Jukic A.M.Z. Weinberg C.R. Baird D.D. Wilcox A.J. Lifestyle and reproductive factors associated with follicular phase length.J. Womens Health (Larchmt). 2007; 16: 1340-1347Crossref PubMed Scopus (41) Google Scholar) and treatments (Kuang et al., 2015Kuang Y. Chen Q. Fu Y. Wang Y. Hong Q. Lyu Q. Ai A. Shoham Z. Medroxyprogesterone acetate is an effective oral alternative for preventing premature luteinizing hormone surges in women undergoing controlled ovarian hyperstimulation for in vitro fertilization.Fertility and sterility. 2015; 104 (62-70.e3)Abstract Full Text Full Text PDF Scopus (153) Google Scholar). These potential confounders should not be simply ruled out.

Background : Over recent years, progestin has been incorporated into the novel ovary stimulation regimens as an alternative for GnRH analogs to prevent premature luteinizing hormone surge, and has become widely used in treatment for infertility. However, the influence of progesterone administration in terms of clinical outcomes and the mechanisms involved remains poorly understood.Methods: The clinic outcomes from patients undergoing ovary stimulation for in vitro fertilization were analyzed. The mouse ovary stimulation model and follicle culture system were used to evaluate the effect of progesterone on oocyte yield, follicle development, granulosa cell proliferation, hormone secretion. Phospho-Specific Protein Microarray, western blot and RT-PCR were employed to explored the signal mechanism.Findings: We present the evidence from patients suggesting that progesterone decreased the oocyte yields, that was rescued by the higher dose of human menopausal gonadotropin. Using both in vivo and in vitro mouse models, we demonstrated that the administration of progesterone inhibited the proliferation of granulosa cells and therefore reduced the growth rate of follicles. The action of progesterone was mediated by progesterone receptor membrane component-1 and rescued by FSH. Using a phosphorylation array, we identified the PI3K/AKT and MAPK are pivotal pathways that integrated the action of progesterone into the FSH signaling network via the downregulation of cyclin D2 and c-fos in granulosa cells.Interpretation: Our findings highlight the mechanism by which progesterone coordinates with FSH to regulate follicle growth and subsequently influence oocyte outcomes in clinic. These findings advance our understanding of the alterations of ovary response to gonadotrophins during progestin primed ovarian stimulation and gives rise to the opportunity for manipulating individual oocyte yields by signaling regulation.Funding Statement: This study was financially supported by the Natural Science Foundation of Shanghai (Grant number 19ZR1429300, awarded to HL), research grants from the National Natural Science Foundation of China (Grant number 81871163, awarded to QL; Grant number 81771533, awarded to YK; Grant number 81671520, awarded to QC), and the National Key Project (Grant number 2018YFC1003000).Declaration of Interests: The authors have declared that no conflict of interest exists.Ethics Approval Statement: Approval for human retrospective analysis was obtained from the institutional Ethics Committee of Shanghai Ninth People’s Hospital affiliated to Shanghai JiaoTong University’s School of Medicine. Animal studies were approved by the institutional Ethics Committees of Care and Use of Experimental Animals of Shanghai Jiaotong University.

旨在利用二代靶向测序技术比较不同靶区对CRISPR/Cas9基因编辑结局的影响,为该技术应用过程中的靶区筛选提供技术参考.本研究进行如下试验:1)以小鼠Pyk2基因为研究对象,针对其第一外显子区设计3个靶区,通过打靶质粒构建、细胞转染及转染细胞DNA二代靶向测序等手段发现,不同靶区总体的基因编辑(Indels)效率相差较大(site1:32.0％;site2:7.9％;site3:69.5％),编辑修复的偏好性也存在较大区别;而对于同一靶区,总编辑效率在2组试验重复中较为稳定(site1:31.8％ vs 32.3％;site2:7.4％vs 8.4％;site3:71.3％vs 67.8％),编辑的偏好性也相对一致;2)针对上述筛出的最佳靶区site1,通过sgRNA与Cas9体外转录、小鼠胚胎显微注射和移植及子代的基因型鉴定等手段,结果发现,上述靶区的基因编辑偏好性在基因编辑动物生产中也得到证实;3)利用上述得到的site1靶区插入一个碱基的突变小鼠,在原靶区位置设计与site1仅相差一个碱基的sgRNA.通过突变小鼠基因组PCR和Cas9酶体外切割试验发现,靶区切割位点单碱基的插入就对该靶区编辑的效率产生显著影响(49.2％vs 0％).综上所述,靶区选择对CRISPR/Cas9基因编辑的结局影响显著,通过二代靶向测序可以有效筛选CRISPR-Cas9基因编辑靶区,并在模式动物生产过程中也获得较好的结果.此外,针对靶区切割位点的单个碱基插入对基因编辑效率影响显著.

With the development of CRISPR/Cas9 system, genetic manipulation of the human germline cell has become a possibility. Whereas, an unspecific activity towards other genome locations, also named off- targets effects, is still a challenge for clinical utility of the CRISPR/Cas9 system in human assisted reproduction. Here, by using animal models, we successfully edit genes both through non-homologous end joining (NHEJ) mediated nucleotides deletion (Ptk2b-8bp) or insertion (Ptk2b+1bp) and homologous recombination mediated precise amino acid site mutation (Ptk2bY402F). Then, these gene editing animal models were screened throughout the whole genome to find the potential off target using Guide-Seq method. On the other hand, To avoid the off target effect of nuclear genome, we also chosen mitochondrial genome as target, which only has a small (∼16.6kb) circular molecule that code 37 genes, and constructed mitochondrial targeted CRISPR/Cas9 system. After applying in cultured cell lines, we found the possibility of this system in mitochondrial genome editing. With the development of CRISPR/Cas9 system, genetic manipulation of the human germline cell has become a possibility. Whereas, an unspecific activity towards other genome locations, also named off- targets effects, is still a challenge for clinical utility of the CRISPR/Cas9 system in human assisted reproduction. Here, by using animal models, we successfully edit genes both through non-homologous end joining (NHEJ) mediated nucleotides deletion (Ptk2b-8bp) or insertion (Ptk2b+1bp) and homologous recombination mediated precise amino acid site mutation (Ptk2bY402F). Then, these gene editing animal models were screened throughout the whole genome to find the potential off target using Guide-Seq method. On the other hand, To avoid the off target effect of nuclear genome, we also chosen mitochondrial genome as target, which only has a small (∼16.6kb) circular molecule that code 37 genes, and constructed mitochondrial targeted CRISPR/Cas9 system. After applying in cultured cell lines, we found the possibility of this system in mitochondrial genome editing.