Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
03 Jan 2014-Science (American Association for the Advancement of Science)-Vol. 343, Iss: 6166, pp 84-87
TL;DR: This work shows that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells, and observes a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation.
Abstract: The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12 , as well as novel hits NF2 , CUL3 , TADA2B , and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
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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 "Genome-Scale CRISPR-Cas9 Knockout S..."
...This approach was also used to identify genes essential for cell viability in cancer and pluripotent stem cells (102)....
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TL;DR: In this paper, the authors describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions, and highlight challenges and future directions.
4,361 citations
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
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
TL;DR: Recently devised sgRNA design rules are used to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results, and a metric to predict off-target sites is developed.
Abstract: CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
2,866 citations
References
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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: 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
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
TL;DR: BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers, with a single substitution (V599E) accounting for 80%.
Abstract: Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
9,785 citations
TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
Abstract: Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
8,197 citations