为了在鸡基因组上对AMHR2基因进行精确编辑,该研究利用crispr.mit.edu:807网站设计并通过PCR退火拟合获得成对的AMHR2基因靶位点,将靶位点分别整合进pX330-U6-Chimeric_dBsa I-CBh-hSpCas9载体骨架,构建靶向AMHR2基因的成对CRISPR/Cas9...为了在鸡基因组上对AMHR2基因进行精确编辑,该研究利用crispr.mit.edu:807网站设计并通过PCR退火拟合获得成对的AMHR2基因靶位点,将靶位点分别整合进pX330-U6-Chimeric_dBsa I-CBh-hSpCas9载体骨架,构建靶向AMHR2基因的成对CRISPR/Cas9敲除载体。将包含成对靶位点的DNA序列整合进pB-CMV-DsRed-CAG-Chimeric.200 bp repeat.Puro-T2A-GFP载体骨架,构建与敲除载体对应的双荧光报告载体。载体测序鉴定正确后分别共转染HEK293T细胞与鸡DF-1细胞,通过细胞荧光粗略判断CRISPR/Cas9系统是否工作。随后,利用Puromycin对基因编辑阳性的DF-1细胞进行药物筛选富集,提取细胞基因组进行TA克隆,进一步检测CRISPR/Cas9系统的工作效率。结果表明靶向鸡AMHR2基因的CRISPR/Cas9敲除系统构建成功,且成对靶位点敲除系统的工作效率高于单一靶位点敲除系统,CRISPR/Cas9敲除系统在DF-1细胞基因组上对AMHR2基因的敲除效率约为60.0%,并在鸡DF-1细胞基因组上实现了AMHR2基因的精确编辑。展开更多
Persistent Müllerian duct syndrome(PMDS)is a rare clinically and genetically overlapping disorder caused by mutations in the anti-Müllerian hormone(AMH)gene or the anti-Müllerian hormone receptor type 2...Persistent Müllerian duct syndrome(PMDS)is a rare clinically and genetically overlapping disorder caused by mutations in the anti-Müllerian hormone(AMH)gene or the anti-Müllerian hormone receptor type 2(AMHR2)gene.Affected individuals present uterus and tubes in normally virilized males and are discovered unexpectedly during other surgeries.Since it is rare and complex,a definitive clinical diagnosis can be missed,and there are no guidelines regarding how to deal with the uterus.In the present study,exome sequencing and Sanger verification were performed for causal variants in 12 PMDS patients.Preoperative diagnoses were made by positive exome sequencing in 8 patients.Of them,7 patients evoked on the basis of ultrasound indicating bilateral testes on the same side of the body.Twelve different AMH variants(2 frameshift/nonsense,1 deletion,8 missense,and 1 in-frame)in 9 patients and 6 different AMHR2 variants(5 missense and 1 splicing)in 3 patients were identified.Seven variants were classified as“pathogenic”or“likely pathogenic”,and 4 of them were novel.All but two patients with AMH defects showed low serum AMH concentrations,but all patients with AMHR2 defects showed elevated AMH levels.During surgery,an abnormal vas deferens was observed in half of the patients.Eight patients underwent orchidopexy with uterine preservation.Of them,2 patients presented complications including irreducible cryptorchidism,and 3 patients developed Müllerian remnant cysts.Three patients underwent subtotal hysterectomy.Of them,one patient had complication of injury to the vas deferens,and one had hemorrhage after operation.This is the first report of PMDS involving a large Chinese population.The present study not only expands the variation spectrum but also provides clinical experience about the management of the uterus.展开更多
文摘为了在鸡基因组上对AMHR2基因进行精确编辑,该研究利用crispr.mit.edu:807网站设计并通过PCR退火拟合获得成对的AMHR2基因靶位点,将靶位点分别整合进pX330-U6-Chimeric_dBsa I-CBh-hSpCas9载体骨架,构建靶向AMHR2基因的成对CRISPR/Cas9敲除载体。将包含成对靶位点的DNA序列整合进pB-CMV-DsRed-CAG-Chimeric.200 bp repeat.Puro-T2A-GFP载体骨架,构建与敲除载体对应的双荧光报告载体。载体测序鉴定正确后分别共转染HEK293T细胞与鸡DF-1细胞,通过细胞荧光粗略判断CRISPR/Cas9系统是否工作。随后,利用Puromycin对基因编辑阳性的DF-1细胞进行药物筛选富集,提取细胞基因组进行TA克隆,进一步检测CRISPR/Cas9系统的工作效率。结果表明靶向鸡AMHR2基因的CRISPR/Cas9敲除系统构建成功,且成对靶位点敲除系统的工作效率高于单一靶位点敲除系统,CRISPR/Cas9敲除系统在DF-1细胞基因组上对AMHR2基因的敲除效率约为60.0%,并在鸡DF-1细胞基因组上实现了AMHR2基因的精确编辑。
基金This study was supported by National Key R&D Program of China(grant No.2018YFC1002702).
文摘Persistent Müllerian duct syndrome(PMDS)is a rare clinically and genetically overlapping disorder caused by mutations in the anti-Müllerian hormone(AMH)gene or the anti-Müllerian hormone receptor type 2(AMHR2)gene.Affected individuals present uterus and tubes in normally virilized males and are discovered unexpectedly during other surgeries.Since it is rare and complex,a definitive clinical diagnosis can be missed,and there are no guidelines regarding how to deal with the uterus.In the present study,exome sequencing and Sanger verification were performed for causal variants in 12 PMDS patients.Preoperative diagnoses were made by positive exome sequencing in 8 patients.Of them,7 patients evoked on the basis of ultrasound indicating bilateral testes on the same side of the body.Twelve different AMH variants(2 frameshift/nonsense,1 deletion,8 missense,and 1 in-frame)in 9 patients and 6 different AMHR2 variants(5 missense and 1 splicing)in 3 patients were identified.Seven variants were classified as“pathogenic”or“likely pathogenic”,and 4 of them were novel.All but two patients with AMH defects showed low serum AMH concentrations,but all patients with AMHR2 defects showed elevated AMH levels.During surgery,an abnormal vas deferens was observed in half of the patients.Eight patients underwent orchidopexy with uterine preservation.Of them,2 patients presented complications including irreducible cryptorchidism,and 3 patients developed Müllerian remnant cysts.Three patients underwent subtotal hysterectomy.Of them,one patient had complication of injury to the vas deferens,and one had hemorrhage after operation.This is the first report of PMDS involving a large Chinese population.The present study not only expands the variation spectrum but also provides clinical experience about the management of the uterus.
