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Hereditas(Beijing) ›› 2018, Vol. 40 ›› Issue (7): 561-571.doi: 10.16288/j.yczz.17-417

• Research Articles • Previous Articles     Next Articles

Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors

Hailong Liu1,Yang Shen1,Yang Gao1,Ling Zhou2,Xiaosong Han1,Changzhi Zhao1,Gaojuan Yang1,Yilong Chen1,Hui Yang1,Shengsong Xie1,2,3()   

  1. 1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
    2. Department of Basic Veterinary Science, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
    3. The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2018-01-23 Revised:2018-04-27 Online:2018-07-20 Published:2018-06-06
  • Supported by:
    Supported by the National Key Research and Development Program of China, Stem Cell and Translational Research(2016YFA0100203);the Fundamental Research Funds for the Central Universities(2662018JC002);the National Transgenic Project of China(2016ZX08006003-004);Students Research Fund(2016048)

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Abstract:

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.

Key words: pig, miRNA, CRISPR/Cas9, sgRNA, knockout