An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations.

Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.

First report of Schistosoma sinensium infecting Tupaia belangeri and Tricula sp. LF.

Schistosoma sinensium belongs to the Asian Schistosoma and is transmitted by freshwater snails of the genus Tricula. Rodents are known definitive hosts of S. sinensium. In 2016, suspected schistosome eggs were found in the feces of the northern tree shrew (Tupaia belangeri) in a field in Lufeng County (latitude, 25°04'50″ N; longitude, 102°19'30″ E; altitude 1820 m), Yunnan Province, China. Morphological analysis suggested that the schistosome was S. sinensium. 18S, 12S and CO1 genes sequencing and phylogenetic analysis showed that this species had the highest similarity to and occupied the same evolutionary branch as S. sinensium from Mianzhu, Sichuan, China. Meanwhile, based on 16S and 28S rDNA sequencing and morphological identification, the snail intermediate host was identified as a species of Tricula, and was found in irrigation channels. Phylogeny indicated that Tricula sp. LF was a sister taxon to T. bambooensis, T. ludongbini. The S. sinensium was able to experimentally infect the captive-bred Tupaia belangeri, and Schistosoma eggs were recovered from all Tupaia belangeri exposed. In this study, we report the infection of Tupaia belangeri and Tricula sp. LF with S. sinensium in Lufeng, Yunnan, southwest China. These findings may improve our understanding of the host range, evolution, distribution, and phylogenetic position of S. sinensium.

Direct-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen.

CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription, and epigenetic modifications in genetic screens. However, it remains challenging to assay the complex molecular readouts after perturbation at high resolution and at scale. By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow.

Establishment of an immortalized intestinal epithelial cell line from tree shrews by lentivirus-mediated hTERT gene transduction.

The intestinal epithelium has an average lifespan of 4-5 days. Normally, primary intestinal epithelial cells can be cultured for about 15 days in vitro. The aim of this study was to explore methods to isolate and immortalize intestinal epithelial cells (IECs) of tree shrews in order to establish a new resource of experimental material and to provide a cell model for drug development and infection mechanism research. Tissue from the small intestine of tree shrews was digested with collagenase XI, neutral protease I, and dithiothreitol. The human telomerase reverse transcriptase gene (hTERT) was transferred into tree shrew IECs using the pHBLV-CMVIE-ZsGreen-Puro vector. The level of hTERT mRNA was detected by quantitative reverse transcription polymerase chain reaction. Immunofluorescence and western blot assays were performed to detect biochemical markers of IECs. The micromorphology of cells was observed with electron microscopy. We then conducted experiments to assess proliferative activity and analyze the karyotype of isolated cells. The results showed the immortalized cell line that we established and screened, maintained the characteristics and biochemical markers of primary IECs. Our results showed that the cell line we established can be considered an alternative cell model for intestinal drug research and for studies on intestinal infection and cell signaling.