基于CRISPR/Cas9技术构建猪miR-136基因编辑载体

Construction of Gig miR-136 Gene Editing Vector Based on CRISPR/Cas9 Technique

为了阐明microRNA对猪产仔数的调节机制,采用CRISPR/Cas9技术构建针对猪miR-136的基因编辑表达载体。通过实验,在miR-136基因成熟区及其下游设计两个单链引导RNA(sgRNA),合成4条磷酸化的单链DNA寡核苷酸,复性后形成两条带粘性末端的双链寡核苷酸片段,插入线性化的载体pX462的BpiI酶切位点处,转化感受态大肠杆菌DH5α,经测序确认,得到两个编辑表达载体pX462-miR-136-1和pX462-miR-136-2。构建了两个编辑miR-136基因的表达载体,为猪繁殖力的调控研究奠定了技术基础。

The present paper aims to explore the molecular mechanism of microRNA on the litter size of pig. The expressed vector to edit the miR-136 gene was constructed using the clustered regularly interspaced short palindromic repeat （CRISPR） and associated nuclease Cas9 （CRISPR/Cas9） technique. A pair of single guide RNAs （sgRNA） were designed at the miR-136 gene mature sequence and its downstream location based on the precursor of pig miR-136. Four single DNA oligonueleotides were synthesized with a phosphorylation on each strand of the 5-end ,and annealed into two double-strand oligo fragments ： oligo 1 and oligo 2. Both of these oligos were inserted into the BpiI site of plasmid pX462, respectively. After transforming into competent DH5α, the sequences of inserts were confirmed for the two recombinant plasmids as pX462-miR- 136-1 and pX462-miR-136-2. Two expression vectors for miR-136 gene editing were constructed, which would be useful for the research of fecundity in pig.