The human genome consists of ~20,000–25,000 protein-coding genes and noncoding regulatory elements including promoters, enhancers, silencers, and insulators. The understanding of how these elements contribute to complex cellular processes remains unclear. Functional genomics attempts to elucidate the relationship between genotypes and phenotypes through gain-of-function, loss-of-function, and mutational assays.
Over the past years, the discovery of the CRISPR/Cas9 system and the development of next-generation sequencing have dramatically accelerated functional genomic studies. Compared to traditional RNAi technology, CRISPR/Cas9 system can completely and permanently deplete protein-coding genes with fewer off-targets. Combining libraries of single-guide RNAs with different CRISPR strategies, CRISPR/Cas9-based pooled screens have been widely used to study gene function at a genome-wide scale since 2014, such as CRISPR/Cas9 knockout (CRISPR KO) screen, CRISPR interference (CRISPRi) screen, CRISPR activation (CRISPRa) screen, Perturb-seq, and base editor screen. Focusing on detailed application scenarios, a variety of sub-libraries targeting interested gene sets, such as metabolic genes, kinome genes, enhancers, and long noncoding RNAs, have been created. CRISPR/Cas9-based screens as powerful tools allow us to discover druggable targets, identify key genes causing drug resistance/sensitivity, explore the mechanisms of cellular signaling and find the genes based on phenotype, etc. Furthermore, novel applications of screening in different models will emerge in the foreseeable future.
Although pooled CRISPR screens are and will further be exploited as a very powerful tool for functional genomic investigations, some issues need to be resolved: (i) The efficiency of sgRNAs. Many existing libraries contain a large proportion of ineffective sgRNAs, some even reach 30%, which will miss some potentially critical genes. (ii) Large-scale pooled libraries limit their application in vivo due to the limitation of cell acquisition. (iii) Novel bioinformatics approaches for analysis of screening data are still required to reduce the false positive hits and allow multi-omics analysis combined with the single-cell transcriptome. (iv) Carefully conducted and well-documented CRISPR screens are important for reproducibility. The current CRISPR screening workflow needs to be standardized.
For the reasons mentioned above, the research topic of this issue aims to present original research, protocols, commentaries, and reviews on methodology or application of functional genomics screening such as:
• Development of new pooled screening strategies, including but not limited to RNAi, CRISPR/Cas9 system
• Optimization of CRISPR/Cas9 screening libraries with high-efficiency sgRNAs
• Minimized whole genome library or sub-libraries targeting specific gene set
• Computational tools or pipeline benchmark for CRISPR screening data
• Novel applications of high-throughput screening in vitro or in vivo
The human genome consists of ~20,000–25,000 protein-coding genes and noncoding regulatory elements including promoters, enhancers, silencers, and insulators. The understanding of how these elements contribute to complex cellular processes remains unclear. Functional genomics attempts to elucidate the relationship between genotypes and phenotypes through gain-of-function, loss-of-function, and mutational assays.
Over the past years, the discovery of the CRISPR/Cas9 system and the development of next-generation sequencing have dramatically accelerated functional genomic studies. Compared to traditional RNAi technology, CRISPR/Cas9 system can completely and permanently deplete protein-coding genes with fewer off-targets. Combining libraries of single-guide RNAs with different CRISPR strategies, CRISPR/Cas9-based pooled screens have been widely used to study gene function at a genome-wide scale since 2014, such as CRISPR/Cas9 knockout (CRISPR KO) screen, CRISPR interference (CRISPRi) screen, CRISPR activation (CRISPRa) screen, Perturb-seq, and base editor screen. Focusing on detailed application scenarios, a variety of sub-libraries targeting interested gene sets, such as metabolic genes, kinome genes, enhancers, and long noncoding RNAs, have been created. CRISPR/Cas9-based screens as powerful tools allow us to discover druggable targets, identify key genes causing drug resistance/sensitivity, explore the mechanisms of cellular signaling and find the genes based on phenotype, etc. Furthermore, novel applications of screening in different models will emerge in the foreseeable future.
Although pooled CRISPR screens are and will further be exploited as a very powerful tool for functional genomic investigations, some issues need to be resolved: (i) The efficiency of sgRNAs. Many existing libraries contain a large proportion of ineffective sgRNAs, some even reach 30%, which will miss some potentially critical genes. (ii) Large-scale pooled libraries limit their application in vivo due to the limitation of cell acquisition. (iii) Novel bioinformatics approaches for analysis of screening data are still required to reduce the false positive hits and allow multi-omics analysis combined with the single-cell transcriptome. (iv) Carefully conducted and well-documented CRISPR screens are important for reproducibility. The current CRISPR screening workflow needs to be standardized.
For the reasons mentioned above, the research topic of this issue aims to present original research, protocols, commentaries, and reviews on methodology or application of functional genomics screening such as:
• Development of new pooled screening strategies, including but not limited to RNAi, CRISPR/Cas9 system
• Optimization of CRISPR/Cas9 screening libraries with high-efficiency sgRNAs
• Minimized whole genome library or sub-libraries targeting specific gene set
• Computational tools or pipeline benchmark for CRISPR screening data
• Novel applications of high-throughput screening in vitro or in vivo