Genetic Screens in Human Cells Using the CRISPR-Cas9 System
01 Dec 2013-
TL;DR: A pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library is described and it is shown that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs.
Abstract: The bacterial CRISPR/Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Finally, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/ sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.
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TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.
4,774 citations
Cites methods from "Genetic Screens in Human Cells Usin..."
...A genomescale lentiviral sgRNA library was developed to generate a pooled loss-of-function genetic screening approach suitable for both positive and negative selection (100, 101)....
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TL;DR: In this paper, Zhang et al. used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify loss-of-function mutations in a melanoma model.
Abstract: Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)–associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi) and have been used to reveal new mechanisms in diverse biological models1-4. Previously, we used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening.
Here, we sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library1, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. To create a new vector capable of producing higher-titer virus (lentiCRISPRv2), we made several modifications, including removal of one of the nuclear localization signals (NLS), human codon-optimization of the remaining NLS and P2A bicistronic linker sequences, and repositioning of the U6-driven sgRNA cassette (Fig. 1a). These changes resulted in a ~10-fold increase in functional viral titer over lentiCRISPRv11 (Fig. 1b).
Figure 1
New lentiviral CRISPR designs produce viruses with higher functional titer.
To further increase viral titer, we also cloned a two-vector system, in which Cas9 (lentiCas9-Blast) and sgRNA (lentiGuide-Puro) are delivered using separate viral vectors with distinct antibiotic selection markers (Fig. 1a). LentiGuide-Puro has a ~100-fold increase in functional viral titer over the original lentiCRISPRv1 (Fig. 1b). Both single and dual-vector systems mediate efficient knock-out of a genomically-integrated copy of EGFP in human cells (Supplementary Fig. 1). Whereas the dual vector system enables generation of Cas9-expressing cell lines which can be subsequently used for screens using lentiGuide-Puro, the single vector lentiCRISPRv2 may be better suited for in vivo or primary cell screening applications.
In addition to the vector improvements, we designed and synthesized new human and mouse GeCKOv2 sgRNA libraries (Supplementary Methods) with several improvements (Table 1): First, for both human and mouse libraries, to target all genes with a uniform number of sgRNAs, we selected 6 sgRNAs per gene distributed over 3-4 constitutively expressed exons. Second, to further minimize off-target genome modification, we improved the calculation of off-target scores based on specificity analysis5. Third, to inactivate microRNAs (miRNAs) which play a key role in transcriptional regulation, we added sgRNAs to direct mutations to the pre-miRNA hairpin structure6. Finally, we targeted ~1000 additional genes not included in the original GeCKO library.
Table 1
Comparison of new GeCKO v2 human and mouse sgRNA libraries with existing CRISPR libraries.
Both libraries, mouse and human, are divided into 2 sub-libraries — containing 3 sgRNAs targeting each gene in the genome, as well as 1000 non-targeting control sgRNAs. Screens can be performed by combining both sub-libraries, yielding 6 sgRNAs per gene, for higher coverage. Alternatively, individual sub-libraries can be used in situations where cell numbers are limiting (eg. primary cells, in vivo screens). The human and mouse libraries have been cloned into lentiCRISPRv2 and into lentiGuide-Puro and deep sequenced to ensure uniform representation (Supplementary Fig. 2, 3). These new lentiCRISPR vectors and human and mouse libraries further improve the GeCKO reagents for diverse screening applications. Reagents are available to the academic community through Addgene and associated protocols, support forums, and computational tools are available via the Zhang lab website (www.genome-engineering.org).
3,833 citations
01 Jun 2014
TL;DR: The development and applications of Cas9 are described for a variety of research or translational applications while highlighting challenges as well as future directions.
Abstract: Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.
3,270 citations
Cites background from "Genetic Screens in Human Cells Usin..."
...selection screens in human cells (Wang et al., 2014; Shalem et al., 2014) by introducing loss-of-function mutations into early, constitutive coding exons of a different gene in each cell (Figure 6F)....
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...…demonstrated the ability to perform robust negative and positive 1272 Cell 157, June 5, 2014 ª2014 Elsevier Inc. selection screens in human cells (Wang et al., 2014; Shalem et al., 2014) by introducing loss-of-function mutations into early, constitutive coding exons of a different gene in each…...
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TL;DR: Long noncoding RNAs (lncRNAs) as discussed by the authors form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome.
Abstract: The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.
3,075 citations
TL;DR: A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells, which will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
Abstract: Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
2,930 citations
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TL;DR: Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
Abstract: As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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TL;DR: The Gene Set Enrichment Analysis (GSEA) method as discussed by the authors focuses on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.
Abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
34,830 citations
TL;DR: To their surprise, it was found that double-stranded RNA was substantially more effective at producing interference than was either strand individually, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
Abstract: Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression Here we investigate the requirements for structure and delivery of the interfering RNA To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference The effects of this interference were evident in both the injected animals and their progeny Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process
15,374 citations
"Genetic Screens in Human Cells Usin..." refers background or methods in this paper
...For all remaining genes, 5-10 candidate sgRNAs were selected with a preference for sequences ordered by (1) the number of matches elsewhere in the genome (2) the number of 1-bp mismatched guide sequences that map elsewhere in the genome (3) the number of transcript models targeted for a given gene (4) the sgRNA score as predicted by the sgRNA efficacy algorithm and (5) the position along the transcript....
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...The target sequences (each encoded by a vector of 80 binary variables representing the presence or absence of each nucleotide (A, C, T, G) at each position (1-20) along the target sequence) of ribosomal protein gene-targeting sgRNAs were used as inputs to the classifier which was trained on the change in abundance observed (encoded by a binary variable corresponding to ‘weak’ and ‘strong’ sgRNAs using a cutoff based on the bimodality of the distribution)....
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TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
12,865 citations
"Genetic Screens in Human Cells Usin..." refers background in this paper
...In this two-component system, a single guide RNA (sgRNA) directs the Cas9 nuclease to cause double-stranded cleavage of matching target DNA sequences (17)....
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TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
12,265 citations