CRISPR/Cas技术能高效进行基因组定点编辑,但不同细菌来源或人工改造的Cas9以及Cpf1等核酸酶识别的PAM (protospacer adjacent motif)有差异,因此不同的基因编辑核酸酶可能采用不同类型的sgRNAs(small guide RNAs)。MicroRNAs (miRNAs)是一类调控性的小分子非编码RNAs,为了研究miRNA前体中是否可能存在特异性高的sgRNAs靶点,本文利用本课题组前期开发的生物信息学软件CRISPR-offinder,对靶向28 645条miRNA前体的11种不同类型sgRNA的丰度及特异性进行了分析,并利用CRISPR/Cas9慢病毒技术构建了猪miR-302/367基因簇敲除细胞系,对构建的猪miRNA敲除细胞系的效率进行了检测。结果表明,每个miRNA前体中平均存在约8种不同类型sgRNA的靶点;通过评估靶向猪miRNA前体sgRNA的脱靶效应,发现其中特异性高的sgRNA仅占18.2%;通过CRISPR/Cas9慢病毒技术成功构建了猪miR-302/367基因簇敲除细胞系,发现通过该技术构建miRNA敲除细胞系的效率为40%。本研究为利用CRISPR/Cas技术靶向敲除miRNA提供了重要资源。

CRISPR/Cas technology enables efficient and specific editing the genome. Since different bacterial sources or artificially modified Cas9, as well as Cpf1 and other nucleases, recognize different PAMs (protospacer adjacent motifs), different gene editing nucleases may use different types of sgRNAs (small guide RNA). MicroRNAs (miRNAs) are a class of regulatory small non-coding RNAs. To determine whether specific targets for sgRNAs in miRNA precursor exit, the abundance and specificity of 11 different types of sgRNA targeting 28 645 miRNA precursors were analyzed in the present study using the CRISPR-offinder, a bioinformatics software developed in our own laboratory. The CRISPR/Cas9 lentivirus technology was used to target the miR-302/367 cluster in a porcine cell line, and its knockout efficiency for the miRNA target was evaluated. The results show that there are about 8 different types of sgRNAs that can target individual miRNA precursors. By assessing the off-target effect, only 18.2% of the sgRNAs showed high specificity for targeting the porcine miRNA precursors. Lastly, using the miR-302/367 cluster target as an example, we showed that the CRISPR/Cas9 lentivirus technology was 40% efficient in successfully establishing correct knockout of the target miRNA in the porcine cell line. This present study provides an important resource for the use of CRISPR/Cas technology to target miRNAs for knockout studies.