Liu Hai Long, Shen Yang, Gao Yang, Zhou Ling, Han Xiao Song, Zhao Chang Zhi, Yang Gao Juan, Chen Yi Long, Yang Hui, Xie Sheng Song
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Department of Basic Veterinary Science, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Yi Chuan. 2018 Jul 20;40(7):561-571. doi: 10.16288/j.yczz.17-417.
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.
CRISPR/Cas技术可实现对基因组的高效且特异性编辑。由于不同的细菌来源或人工改造的Cas9，以及Cpf1和其他核酸酶识别不同的原间隔序列临近基序（PAM），不同的基因编辑核酸酶可能会使用不同类型的小向导RNA（sgRNA）。微小RNA（miRNA）是一类具有调控作用的小非编码RNA。为了确定miRNA前体中sgRNA的特异性靶点是否存在，本研究使用我们自己实验室开发的生物信息学软件CRISPR-offinder，分析了针对28645个miRNA前体的11种不同类型sgRNA的丰度和特异性。利用CRISPR/Cas9慢病毒技术靶向猪细胞系中的miR-302/367簇，并评估其对miRNA靶点的敲除效率。结果表明，约有8种不同类型的sgRNA可靶向单个miRNA前体。通过评估脱靶效应，只有18.2%的sgRNA对靶向猪miRNA前体具有高特异性。最后，以miR-302/367簇靶点为例，我们表明CRISPR/Cas9慢病毒技术在猪细胞系中成功建立目标miRNA正确敲除的效率为40%。本研究为利用CRISPR/Cas技术靶向miRNA进行敲除研究提供了重要资源。
