Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem–loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.

Dairy products are a significant source of iodine, and their contribution to iodine intake must be evaluated regularly. However, there is a lack of data on iodine intake from dairy products in China. Through a cross-sectional study, we determined the iodine content of dairy products in the Chinese diet and estimated iodine intake among Chinese children. Intake records for 30 consecutive days were used to investigate the consumption of dairy products by 2009 children from Yunnan and Liaoning Provinces. The iodine contents of 266 dairy products with high intake frequency were determined using inductively coupled plasma–mass spectrometry (ICP-MS). We then calculated the iodine intake and contribution of dairy products and explored the related factors of dairy iodine intake through a generalized linear mixed model. Ultra-high-temperature (UHT) sterilized milk accounted for 78.7% of the total dairy products, with an iodine content of 23.0 μg/100 g. The dairy product intake rate of children in China was 83.6%, with an average daily intake of 143.1 g. The median iodine intake from milk and dairy was 26.8 μg/d, 41.5% of the estimated average recommendation (EAR) for younger children and 31.8% of the EAR for older children. The daily milk iodine intake of children in Yunnan Province was 9.448 μg/day lower than that of children in Liaoning Province (p < 0.001), and the daily iodine intake of children in rural areas was 17.958 μg/day lower than that of children in urban areas (p < 0.001). Chinese dairy products were rich in iodine, and the content of iodine was intermediate to that reported in Europe and the USA. However, children’s daily intake of milk iodine was lower than that of children in other developed countries due to the lower daily intake of dairy products, especially those in rural areas.

Liang Y, et al. J Vet Sci. 2022 Oct;23:e90. https://doi.org/10.4142/jvs.22088

Transcription factors play important roles in the growth and development of various tissues in pigs, such as muscle, fat, and bone. A transcription-factor-scale activation library based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system could facilitate the discovery and functional characterization of the transcription genes involved in a specific gene network. Here, we have designed and constructed a CRISPR activation (CRISPRa) sgRNA library, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. The sgRNA library, including sgRNA with MS2 loops, is a single-vector system and is packaged with lentivirus for cell screening. Porcine PK15 cells expressing the porcine OCT4 promoter driving EGFP, dCas9 fused with VP64, and MS2-binding protein-p65-HSF1 were constructed, and then, the sgRNA activation library was used to screen the transcription factors regulating OCT4 expression. After the lentiviral transduction and deep sequencing of the CRISPR sgRNAs library, the highest ranking candidate genes were identified, including 31 transcription factors activating OCT4 gene expression and 5 transcription factors inhibiting OCT4 gene expression. The function and gene regulation of the candidate genes were further confirmed by the CRISPR activation system in PK15 cells. The CRISPR activation library targeting pig transcription factors provides a promising platform for the systematic discovery and study of genes that determine cell fate.