Quartet RNA reference materials improve the quality of transcriptomic data through ratio-based profiling.

Certified RNA reference materials are indispensable for assessing the reliability of RNA sequencing to detect intrinsically small biological differences in clinical settings, such as molecular subtyping of diseases. As part of the Quartet Project for quality control and data integration of multi-omics profiling, we established four RNA reference materials derived from immortalized B-lymphoblastoid cell lines from four members of a monozygotic twin family. Additionally, we constructed ratio-based transcriptome-wide reference datasets between two samples, providing cross-platform and cross-laboratory 'ground truth'. Investigation of the intrinsically subtle biological differences among the Quartet samples enables sensitive assessment of cross-batch integration of transcriptomic measurements at the ratio level. The Quartet RNA reference materials, combined with the ratio-based reference datasets, can serve as unique resources for assessing and improving the quality of transcriptomic data in clinical and biological settings.

Multi-omics data integration using ratio-based quantitative profiling with Quartet reference materials.

Characterization and integration of the genome, epigenome, transcriptome, proteome and metabolome of different datasets is difficult owing to a lack of ground truth. Here we develop and characterize suites of publicly available multi-omics reference materials of matched DNA, RNA, protein and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters. These references provide built-in truth defined by relationships among the family members and the information flow from DNA to RNA to protein. We demonstrate how using a ratio-based profiling approach that scales the absolute feature values of a study sample relative to those of a concurrently measured common reference sample produces reproducible and comparable data suitable for integration across batches, labs, platforms and omics types. Our study identifies reference-free 'absolute' feature quantification as the root cause of irreproducibility in multi-omics measurement and data integration and establishes the advantages of ratio-based multi-omics profiling with common reference materials.

Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing.

The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.

Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9.

Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

Lcn5 promoter directs the region-specific expression of cre recombinase in caput epididymidis of transgenic mice.

Epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. To study the function of genes in the caput epididymidis using the Cre/loxP system, we generated Lcn5-Cre transgenic mice in which the expression of Cre recombinase is driven by the 1.8-kb Lcn5 promoter. A total of 11 founder mice carrying the Lcn5-Cre transgene were identified by PCR from 38 offspring, and the integration efficiency was 28.9%. However, only 1 of the 11 transgenic mouse lines were revealed with the Cre recombinase expressed specifically in caput epididymidis. Furthermore, expression of Cre mRNA was first observed on Postnatal Day 30 and continued to increase during development. Subsequently, Cre protein distribution was assessed by crossing Lcn5-Cre transgenic mice into the mT/mG reporter line. As expected, the Cre recombinase activity was only found in principal cells of the middle/distal caput epididymidis. The tissue-specific expression of Cre protein in the caput epididymidis was further confirmed using Lcn5-Cre mice crossed with a mouse strain carrying Aip1 conditional alleles (Aip1(flox/+)). In summary, a transgenic mouse line expressing Cre recombinase in principal cells of caput epididymidis was established. This transgenic mouse line can be used to generate conditional, caput epididymidis-specific knockout mouse models by crossing with mice harboring floxed (LoxP flanked) genes.