Massively parallel high-order combinatorial genetics in human cells

Abstract

The systematic functional analysis of combinatorial genetics has been limited by the throughput that can be achieved and the order of complexity that can be studied. To enable massively parallel characterization of genetic combinations in human cells, we developed a technology for rapid, scalable assembly of high-order barcoded combinatorial genetic libraries that can be quantified with high-throughput sequencing. We applied this technology, combinatorial genetics en masse (CombiGEM), to create high-coverage libraries of 1,521 two-wise and 51,770 three-wise barcoded combinations of 39 human microRNA (miRNA) precursors. We identified miRNA combinations that synergistically sensitize drug-resistant cancer cells to chemotherapy and/or inhibit cancer cell proliferation, providing insights into complex miRNA networks. More broadly, our method will enable high-throughput profiling of multifactorial genetic combinations that regulate phenotypes of relevance to biomedicine, biotechnology and basic science.Complex biological traits are regulated by the concerted action of combinatorial gene sets 1 . For example, multiple genetic factors are needed to reprogram somatic cells into induced pluripotent stem cells or distinct cell types 2 . Combinatorial drug therapies can achieve enhanced efficacy over conventional monotherapies, because targeting multiple pathways can be synergistic 3 . Furthermore, although genomewide association studies have implicated multiple individual loci in multifactorial human diseases, these loci can explain only a minor Reprints and permissions information is available online at

Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity

“…In contrast to typical targeted CRISPR screens, in which gRNAs are designed to modulate individual genes, PRISM does not require any assumptions regarding potential targets and enables unbiased and high-level perturbations of cellular networks. Thus, randomized gRNA screening with PRISM is complementary to targeted screening strategies that have been used with CRISPR-Cas nucleases, crisprTFs, and RNA interference (Blancafort et al, 2005; Demir and Boutros, 2012; Gilbert et al, 2014; Konermann et al, 2015; Moffat et al, 2006; Park et al, 2003; Parnas et al, 2015; Paulsen et al, 2009; Root et al, 2006; Santos and Stephanopoulos, 2008; Shalem et al, 2014; Wang et al, 2014; Whitehurst et al, 2007; Wong et al, 2015; Wong et al, 2016). Randomized gRNA screening with crisprTFs involves a simple library construction procedure and enables global perturbations of transcriptional networks that might not be accessible by traditional single- or multiple-gene perturbations.…”

“…For example, crisprTF-based platforms enable bi-directional gene activation and repression in eukaryotic systems (Chavez et al, 2015; Farzadfard et al, 2013; Gilbert et al, 2013; Mali et al, 2013; Nishimasu et al, 2014; Zalatan et al, 2015) and have been used for genome-wide targeted screens owing to the ease of designing and synthesizing guide RNAs (gRNAs) (Gilbert et al, 2014; Horlbeck et al, 2016; Konermann et al, 2015). In addition, strategies for higher-order perturbations using barcoded combinatorial genetic screens in human cells have been adapted to be compatible with CRISPR-Cas9 screens (Wong et al, 2015; Wong et al, 2016). Existing CRISPR-Cas9-based screening strategies rely on gRNAs designed to target individual genes while minimizing off-target effects (Cencic et al, 2014; Frock et al, 2015; Gilbert et al, 2014; O'Geen et al, 2015; Parnas et al, 2015; Shalem et al, 2014; Wang et al, 2014; Wu et al, 2014).…”

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

“…Despite its simplicity for multiplexed genetic perturbations (10)(11)(12), new methods are needed to enable highthroughput CRISPR-Cas9-based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13,14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays.…”

High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

“…Lentiviral-based library screening is the most commonly used method for high-throughput genetic screening [41][42][43] . In this method, diversified genetic elements are cloned into a lentiviral carrier plasmid and transfected into a virus-producing cell line with packaging and envelope plasmids to produce a lentiviral library, which is then used to infect the cells of interest.…”