scholarly journals Genetic Screens in Human Cells Using the CRISPR-Cas9 System

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 80-84 ◽  
Author(s):  
Tim Wang ◽  
Jenny J. Wei ◽  
David M. Sabatini ◽  
Eric S. Lander
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Negative Selection ◽  
Topoisomerase Ii ◽  
Massively Parallel Sequencing ◽  
Sequence Motifs ◽  
Loss Of Function ◽  
Specific Sequence ◽  
Guide Rna ◽  
A Genome

The bacterial clustered regularly interspaced short palindromic repeats (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, whereas 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. Last, 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.

scholarly journals Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

2019 ◽  
Author(s):  
Christopher J Giuliano ◽  
Ann Lin ◽  
Jason Sheltzer
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Protein Function ◽  
Mammalian Cells ◽  
Gene Loss ◽  
Loss Of Function ◽  
Guide Rna ◽  
Mammalian Cell Lines ◽  
Clonal Cell Lines ◽  
Rna Design

CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.

scholarly journals Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

2019 ◽  
Author(s):  
Christopher J Giuliano ◽  
Ann Lin ◽  
Jason Sheltzer
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Protein Function ◽  
Mammalian Cells ◽  
Gene Loss ◽  
Loss Of Function ◽  
Guide Rna ◽  
Mammalian Cell Lines ◽  
Clonal Cell Lines ◽  
Rna Design

CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals The Potential Use of the CRISPR-Cas System for HIV-1 Gene Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Gabriela De Nardi Sanches-da-Silva ◽  
Luiza Fonseca Sales Medeiros ◽  
Fabio Mitsuo Lima
Keyword(s):  
Gene Therapy ◽  
Current Knowledge ◽  
Genetic Material ◽  
Specific Gene ◽  
Loss Of Function ◽  
Complementary Sequence ◽  
Guide Rna ◽  
Ethical Implications ◽  
A Genome ◽  
Hiv 1

The HIV-1 virus (human immunodeficiency virus) affects 36.9 million people worldwide, with approximately 900000 deaths in 2017. The virus carrier can develop severe immunodeficiency since CD4+ T lymphocytes are the main target, leading to acquired immunodeficiency syndrome (AIDS). Despite advances in pharmacological treatment, it is still difficult to eliminate latent reservoirs, becoming one of the main obstacles for viral eradication. The CRISPR- (clustered regularly interspaced short palindromic repeat-) Cas system is a genome-editing method which uses a guide RNA, a complementary sequence to the interested site, recruiting a nuclease that can break the viral or the host cell genetic material. From this double-stranded break, cellular repair mechanisms are activated being able to generate deletions, insertions, or substitutions, in order to inactivate specific gene loci, leading to loss of function. The objective of this minireview is to synthesize the current knowledge on the application of CRISPR-Cas-based gene therapy for HIV-1. The strategies encompass all steps of the viral infection cycle, from inhibition of cell invasion, through viral replication and integration inhibition, to excision of the latent provirus. Off-target effects and ethical implications were also discussed to evaluate the safety of the approach and viability of its application in humans, respectively. Although preclinical and clinical tests are still needed, the recent results establish an exciting possibility of applying this technology for prophylaxis and treatment of HIV-1.

scholarly journals Long Inverted Repeats Are an At-Risk Motif for Recombination in Mammalian Cells

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1873-1883 ◽  
Author(s):  
Alan S Waldman ◽  
Hiep Tran ◽  
Edie C Goldsmith ◽  
Michael A Resnick
Keyword(s):  
At Risk ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Direct Repeat ◽  
Fold Increase ◽  
Inverted Repeats ◽  
Sequence Motifs ◽  
Common Motif ◽  
Simplex Virus

Abstract Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk− fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10−5 events per cell per generation. Of the tk+ segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk+ segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background: The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.Results: We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.Conclusions: This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Interaction between DNA damage response, translation and apoptosome determines cancer susceptibility to TOP2 poisons

2019 ◽  
Author(s):  
Chidiebere U Awah ◽  
Li Chen ◽  
Mukesh Bansal ◽  
Aayushi Mahajan ◽  
Jan Winter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Cancer Susceptibility ◽  
Protein Subunit ◽  
Ribosomal Subunits ◽  
Topoisomerase Ii Poisons ◽  
Etoposide Treatment ◽  
A Genome ◽  
Genome Scale

AbstractTopoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We show that glioblastoma (GBM), the most malignant of all primary brain tumors in adults is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 impaired the induction of pro-apoptotic gene APAF1 following etoposide treatment, and led to resistance to this drug and doxorubicin. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from multiple cancers.Graphical Abstract

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. Results We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. Conclusions This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

2021 ◽  
Vol 2 ◽  
Author(s):  
Daniel Allen ◽  
Michael Rosenberg ◽  
Ayal Hendel
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Nuclear Staining ◽  
Two Component System ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Therapeutic Benefits ◽  
A Genome ◽  
Cas9 Protein

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

scholarly journals Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

2018 ◽  
Author(s):  
Byungjin Hwang ◽  
Wookjae Lee ◽  
Soo-Young Yum ◽  
Yujin Jeon ◽  
Namjin Cho ◽  
...  
Keyword(s):  
Cell Fate ◽  
Mammalian Cells ◽  
Cytidine Deaminase ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Targeted Mutagenesis ◽  
Guide Rna ◽  
A Genome ◽  
Organismal Development ◽  
And Function

ABSTRACTDetermining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods have provided new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we developed a genome editing strategy using a cytidine deaminase fused with inactive Cas9 (dCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns served as a genetic barcode, which was induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.

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