National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.
Biotechnol J. 2022 Jul;17(7):e2100408. doi: 10.1002/biot.202100408. Epub 2021 Nov 10.
As an important farm animal, pig functional genomic study can help understand the molecular mechanism related to the key economic traits of pig, such as growth, reproduction, or disease. The genome-scale library based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system facilitates discovery of key genes involved in a specific function or phenotype, allowing for an effective "phenotype-to-genotype" strategy for functional genomic study.
We designed and constructed a pig genome-scale CRISPR/Cas9 knockout library targeting 16,888 genes with 970,001 unique sgRNAs. The library is a single-vector system including both Cas9 and sgRNA, and packaged into lentivirus for an easy cell delivery for screening. To establish a screening method in pig cells, we used diphtheria toxin (DT)-induced cell death as a model to screen the host genes critical for DT toxicity in pig PK-15 cells. After lentiviral transduction and two sequential screening with DT treatment, the highest-ranking candidates we identified were previously validated genes, HBEGF, DPH1, DPH2, DPH3, DPH5, DNAJC24, and ZBTB17, which are DT receptor and the key factors involved in biosynthesis of diphthamide, the target of DT action. The function and gene essentiality of candidates were further confirmed by gene knockout and DT toxicity assay in PK-15 cells.
Our CRISPR knockout library targeting pig whole genome establishes a promising platform for pig functional genomic analysis.
作为一种重要的家畜动物，猪的功能基因组研究有助于理解与猪生长、繁殖或疾病等关键经济性状相关的分子机制。基于成簇规律间隔短回文重复（CRISPR）/CRISPR 相关内切酶 Cas9（Cas9）系统的基因组规模文库有助于发现涉及特定功能或表型的关键基因，实现了有效的“表型到基因型”功能基因组研究策略。
我们设计并构建了一个针对 16888 个基因的猪基因组规模的 CRISPR/Cas9 敲除文库，该文库包含 970011 个独特的 sgRNA。该文库是一个包含 Cas9 和 sgRNA 的单载体系统，包装成慢病毒，便于细胞转染进行筛选。为了在猪细胞中建立筛选方法，我们使用白喉毒素（DT）诱导的细胞死亡作为模型，筛选在猪 PK-15 细胞中对 DT 毒性至关重要的宿主基因。经过慢病毒转导和两次 DT 处理的连续筛选后，我们确定的排名最高的候选基因是先前验证过的基因，包括 HBEGF、DPH1、DPH2、DPH3、DPH5、DNAJC24 和 ZBTB17，它们是 DT 受体和 diphthamide 生物合成的关键因子，是 DT 作用的靶标。通过在 PK-15 细胞中进行基因敲除和 DT 毒性测定，进一步证实了候选基因的功能和基因必需性。
我们针对猪全基因组的 CRISPR 敲除文库为猪功能基因组分析建立了一个有前途的平台。
