بررسی ویرایش ژن dazl در ژنوم رویان بز بختیاری با استفاده از crispr/cas9 و تکنیک های انتقال هسته سلول سوماتیک

Crispr/cas9 در حال ایجاد تغییرات در مهندسی ژنوم و ویژگی های مختلف دام به ویژه صفات اقتصادی است. dazl یک پروتئین حفاظت شده متصل به rna است که برای تمایز و توسعه سلول های زایا، ضروری است. نرهای ناک اوت شده برای این ژن، قادر به تولید اسپرم نیستند. هدف از این مطالعه، ایجاد رویان بز ناک اوت برای ژن dazl بود. با استفاده از grna طراحی شده در وکتور px459، پلاسمیدها ساخته و کلون شدند. پلاسمید نوترکیب با استفاده از روش الکتروپوریشن به سلول های فیبروبلاست منتقل شده و سلول های ترانسفکت شده با تیمار پورمایسین انتخاب شدند. سلول‌های ویرایش شده از مسیر روند انتقال هسته سلول سوماتیک (شبیه سازی)، به تخمک‌های بدون هسته متصل شده و سپس، همجوشی الکتریکی غشای سلولی اعمال شد. رویان های حاصل از روند شبیه سازی از نظر مراحل رویانی و رسیدن به مراحل تسهیم و بلاستوسیست بررسی شدند. نتایج بررسی بیان ژن، موفقیت تکنیک crispr/cas9 برای سرکوب ژن dazl را نشان داد. نرخ تکوین بلاستوسیت حاصل از روند شبیه سازی با سلول های ناک اوت شده، تفاوت قابل توجهی با گروه شاهد نداشت. با کمک بررسی توالی یابی ژنتیکی، میزان ناک اوت ژن dazl در سلول فیبروبلاست، 83.3 و در بلاستوسیست‌های حاصل از شبیه سازی، 55.3 درصد به دست آمد. جهش ها از نوع indel و همگی به شکل تغییر قالب خوانش پروتئین بودند که منجر به تغییر پروتئین می شود. بلاستوسیت‌های تولید شده را می توان برای انتقال به حیوان گیرنده و تولید بزهای ناک اوت شده برای ژن dazl برای مطالعات بیشتر و بهینه ‌سازی پیوند سلول های بنیادی اسپرماتوگونیال استفاده کرد.

investigating dazl gene editing in goat embryos using the crispr/cas9 system and somatic cell nuclear transfer techniques

introduction: the crispr/cas9 (clustered regularly interspaced palindromic repeats/crispr-associated protein9) method can create a nucleotide sequence complementary to the target sequence in the desired gene by guide-rna (grna) together with the cas9 protein, which is a cutting enzyme for cutting in the both dna strands of the desired sequence accurately and clearly. the dazl (deleted in azoospermia-like) gene encodes potential rna-binding proteins that are expressed in male and female germ cells before and after birth. dazl, which acts by post-transcriptionally binding mrna in 3’ untranslated regions, regulates the germ cell cycle. dazl initiates the sexual differentiation of embryonic germ cells. the transfer and transplantation of gene-edited germ cells into recipient males is an effective method for targeted mutagenesis engineering. in mice knocked out for the dazl gene, the number of testicular stem cells was reduced and it was found that the dazl gene plays an important role in the differentiation of spermatogonial cells. the purpose of the current research is to edit the dazl gene by knocking it out using the crispr/cas9 technique and transferring the somatic cell nucleus into the genome of the bakhtiari goat embryo. the inactivation of the gene will be investigated both at the level of the embryonic cell and the resulting embryo. there are no reports on the production of bakhtiari goat cells and embryos edited for the dazl gene by crispr technique and somatic nuclear transfer to improve any traits, including reproductive ones.materials and methods: to target the dazl gene and explore (predict) potential off-target genomic sites, guide rna (20 bp sequences) immediately upstream of each 5′-ngg in the dazl gene was designed using the choopchoop tool. plasmid px459 (9151 bp) was used to insert the grna sequence into the cas9 vector and determine the characteristics and replication of the vector. this plasmid encodes the cas9 protein along with the puromycin resistance gene under the promoter/enhancer, caggs, as well as the grna scaffold under the u6 promoter. plasmid px459 was digested by bbsi-hf (neb #r3539) at 37°c for 10 min, followed by purification by nucleospin gel and pcr clean-up midi kit (#740,986.20, machery/nagel). the purified piece was kept at -20°c for later use. ligation of oligoduplex carrying diluted grna sequence (1:20 ratio from 10 μm source) (1 μl), digested px459 vector (50 ng), 10x t4dna ligase buffer (2 μl), and t4dna ligase (1 μl) in the final concentration of 20 μl of the reaction was carried out. ligation mixture transformation was performed with neb 5-alpha competent e. coli (#c2987i) and then placed on agar culture medium with 100 μg/ml ampicillin and incubated at 37°c overnight. from the cultured plate, five colonies were selected and each was cultured in lb culture medium, followed by miniprep plasmid extraction (genejet plasmid miniprep kit, #k0502). plasmids carrying the crispr system were transferred into cells through an electroporation system.results and discussion: the present results proved the possibility of knocking out the dzal gene using the crispr/cas9 technique in both fibroblast cell lines and bakhtiari goat embryos. we performed somatic cell nuclear transfer (scnt) to examine the embryonic developmental capacity to transition to the cleavage and blastocyst stages. the embryos created by knocking out the dazl gene could grow and develop. the sequencing analysis using decodr (deconvolution of complex dna repair) software showed the knock-out rate of the dazl gene in fibroblast cells was 83.3% and in cloned blastocysts, it was 55.3%. the rate of blastocyst formation resulting from the cloning process with knockout cells was not significantly different from the control group. based on these studies, we decided to edit the dazl gene to produce knockout goat embryos by scnt. the embryos were examined and evaluated with the help of pcr-rflp test and sequencing. it turned out that in the first iteration, a knockout of 60.66% (the first ten embryos were paid), and in the second iteration, a knockout of 49.99% (the second ten embryos were paid) were achieved. such mutations have not been described or detected in iranian bakhtiari goats, although polymorphisms have been identified. using genetic sequencing, the mutations were of the indel type and were all in the form of frameshift, which resulted in a change in the protein.conclusions: the crispr/cas9 system easily influences the desired gene and can be used as a strategy to produce livestock animals that are superior in milk and meat production, reproduction, quality, disease resistance, etc. this research demonstrated the possibility of gene editing using the crispr/cas9 technique in fibroblast cell lines and bakhtiari goat embryos. the blastocytes produced can be used for transfer to the recipient animal and production of dazl gene knockout goats for further study and optimization of spermatogonial stem cell transplantation.