RNA helicase D1PAS1 resolves R-loops and forms a complex for mouse pachytene piRNA biogenesis required for male fertility

To investigate the function of RNA helicase D1PAS1 in mouse spermatogenesis, we established D1Pas1 knockout (KO) and D1Pas1-FLAG knockin (KI) lines in which the D1Pas1 gene has been knocked out by deleting the transcription start site and the 3XFLAG sequences added at the end of C-term by CRISPR/Cas9. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different biological replicates at three different developmental stages. Investigated small RNA abundance from WT and KO testis via small RNA-seq. To profile the genome-wide D1PAS1 RNA helicase dependent R-loop loci, we performed BisMapR sequencing analysis with D1PAS1 KI and KO spermatocytes. Expression profiling of differentially expressed genes by RNA-seq at three different developmental stages, small RNA abundance by small RNA-seq, genome-wide RNA:DNA hybrids (R-loop) profiling by BisMapR, and D1PAS1-bound RNA by eCLIP-sequencing.为探究RNA解旋酶（RNA helicase）D1PAS1在小鼠精子发生中的功能，我们通过CRISPR/Cas9技术敲除D1Pas1基因的转录起始位点，并在其C端末端插入3×FLAG序列，成功构建了D1Pas1基因敲除（knockout, KO）与D1Pas1-FLAG敲入（knockin, KI）工程小鼠系。随后，我们针对三个不同发育阶段的3份生物学重复样本的RNA测序（RNA-seq）数据开展基因表达谱分析，并通过小RNA测序（small RNA-seq）检测野生型（wild type, WT）与KO小鼠睾丸中的小RNA表达丰度。为解析全基因组范围内依赖D1PAS1 RNA解旋酶的R环（R-loop）位点分布，我们以D1PAS1 KI与KO精母细胞为实验材料，采用BisMapR测序技术进行分析。本研究获取的多组学数据涵盖：三个发育阶段的差异表达基因RNA-seq表达谱、睾丸小RNA测序丰度数据、全基因组RNA:DNA杂交链（R环）的BisMapR测序图谱，以及通过增强型交联免疫沉淀测序（enhanced cross-linking immunoprecipitation sequencing, eCLIP-seq）得到的D1PAS1结合RNA数据集。