The interrogation of gene function is an overarching subject in life science research. In recent years, genome editing technology represented by the CRISPR-Cas9 system has enabled functional screening of genes directly in mammalian organisms (Shalem, et al. Science 2014; Wang, et al. Science 2014; Koike-Yusa, et al. Nature Biotechnol 2014; Zhou et al, Nature 2014). However, protein-coding genes account for less than 3% of the genome. Researchers have found that more and more non-coding elements play an essential role in biological processes, such as non-coding RNA, especially long non-coding RNA (lncRNA). lncRNA abnormalities are related to the occurrence and development of many diseases such as cancer. Unfortunately, among the more than 20,000 lncRNAs that have been annotated, the function of most lncRNAs is unknown. How to achieve functional screening of such genomic elements has become a research field of interest.

Genome editing technologies are able to achieve knockout of protein-coding genes through single-point cutting of the target region, introducing small fragment insertions or deletions (indels) to cause damage to the reading frame of gene translation. However, this method is not feasible for non-coding elements that are independent of the reading frame. Although there are some reports of saturation screening using sgRNA libraries to study the regulatory elements of a single or a small number of genes (Canver MC et al., Nature, 2015; Neville E. Sanjana et al., Science, 2016), effective technologies to perform genome-scale screening of non-coding components are still missing. 

Wensheng Wei's group from Peking University, collaborated with Shirley Liu's group from Harvard University, established a paired-guide RNA (pgRNA) library construction method. They disrupted the expression and function of lncRNA through large fragment deletion and achieved functional screenings in multiple cancer cell lines through lentiviral delivery. lncRNAs that positively and negatively regulate cancer cell proliferation were successfully identified from a CRISPR library of ~ 12,000 pgRNA. The function of candidate lncRNAs was verified through a variety of genetic methods. They also explored the mechanism of action of these genes through bioinformatics analysis and expression profiling. Interestingly, by analyzing the expression levels of candidate lncRNAs at different stages of tumor cell development, it is found that the identified lncRNAs that positively regulate cell proliferation play a carcinogenic effect, while the lncRNAs that negatively regulate cell proliferation play a tumor suppressor role. It is the first time to achieve genome-scale functional screening for non-coding elements. The establishment of this high-throughput technology platform not only helps people study non-coding elements that affect cell proliferation but also offers a tool to explore non-coding elements or unannotated regions in the genome that play other important roles.

The manuscript was published online on October 31 in Nature Biotechnology (Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library). Shiyou Zhu, a doctoral student in the School of Life Sciences, Peking University (PTN12), and Wei Li, a postdoctoral fellow in the Harvard School of Public Health, are the co-first authors of the paper. Professors Wensheng Wei and Xiaole Shirley Liu are the co-corresponding authors. The project was supported by funds from the National Science Foundation of China, Beijing Advanced Innovation Center for Genomics at Peking University, the Peking-Tsinghua Center for Life Sciences (to Wensheng Wei), the NIH grant (to Xiaole Shirley Liu and Myles Brown), and the Claudia Adams Barr Award in Innovative Basic Cancer Research from the Dana-Farber Cancer Institute.Construction of CRISPR large fragment deletion library and high-throughput functional screening of lncRNA.

Construction of CRISPR large fragment deletion library and high-throughput functional screening of lncRNA.

(a) pgRNA library cloning construction method. (b) The process of pgRNA library screening. (c) Candidate genes obtained by negative selection. (d) Candidate genes obtained through positive selection.