A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Hultquist, Judd F.; Schumann, Kathrin; Woo, Jonathan M.; Manganaro, Lara; McGregor, Michael; Doudna, Jennifer A.; Simon, Viviana; Krogan, Nevan J.; Marson, Alexander

doi:10.1016/j.celrep.2016.09.080

Cited by 169 publications

(213 citation statements)

References 87 publications

(126 reference statements)

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“…Similarly, phenotypes can be observed with synthetic crRNAs targeting essential genes that would be lost in long-term pooled CRISPR screens. A very recent study also supports our conclusions by demonstrating the utility of complexing synthetic crRNAs with recombinant Cas9 protein, and introducing the resulting ribonucleoproteins into primary T cells via electroporation[29]. This method was able to achieve functional knockout of control genes in roughly two thirds of transfected cell populations, and was subsequently used for the screening of 45 genes in 96 well format.…”

Section: Discussionsupporting

confidence: 74%

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Tan¹,

Martin²

2016

PLoS ONE

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To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.

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Section: Discussionsupporting

confidence: 74%

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Tan¹,

Martin²

2016

PLoS ONE

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“…However, some cells are difficult to transfect using liposomes. These problems could be solved by electroporation, especially using an electroporator equipped with multiwell modules, as reported by Hultquist et al (2016). We validated the genes using original and newly designed sgRNAs.…”

Section: Discussionmentioning

confidence: 92%

“…Some groups have previously attempted to develop arrayed CRISPR screens (Hultquist et al 2016;McCleland et al 2016;Tan and Martin 2016;Datlinger et al 2017;Strezoska et al 2017). These studies were based on guide RNA libraries consisting of individual lentiviral sgRNAs for single genes or synthesized CRISPR RNA (crRNA) in a manner similar to siRNA screens.…”

mentioning

confidence: 99%

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Lee

Kim

et al. 2018

Genome Res.

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Pooled CRISPR screens based on lentiviral systems have been widely applied to identify the effect of gene knockout on cellular phenotype. Although many screens were successful, they also have the limitation that genes conferring mild phenotypes or those essential for growth can be overlooked, as every genetic perturbation is incorporated in the same population. Arrayed screens, on the other hand, incorporate a single genetic perturbation in each well and could overcome these limitations. However, arrayed screens based on siRNA-mediated knockdown were recently criticized for low reproducibility caused by incomplete inhibition of gene expression. To overcome these limitations, we developed a novel arrayed CRISPR screen based on a plasmid library expressing a single guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4542 sgRNAs (three sgRNAs per gene). This screen revealed host factors required for infection by coxsackievirus B3 (CVB3) from Picornaviridae, which includes human pathogens causing diverse diseases. Many host factors that had been overlooked in a conventional pooled screen were identified for CVB3 infection, including entry-related factors, translational initiation factors, and several replication factors with different functions, demonstrating the advantage of the arrayed screen. This screen was quite reliable and reproducible, as most genes identified in the primary screen were confirmed in secondary screens. Moreover, ACBD3, whose phenotype was not affected by siRNA-mediated knockdown, was reliably identified. We propose that arrayed CRISPR screens based on sgRNA plasmid libraries are powerful tools for arrayed genetic screening and applicable to larger-scale screens.

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“…For example, a naturally occurring deletion in the CCR5 gene encoding an essential coreceptor of HIV confers resistance to HIV infection, providing the basis of cell therapy against HIV infection (Hendel et al 2015;Hultquist et al 2016;Xu et al 2017). …”

Section: Discussionmentioning

confidence: 99%

CRISPR RNAs trigger innate immune responses in human cells

et al. 2018

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Here, we report that CRISPR guide RNAs (gRNAs) with a 5 ′ -triphosphate group (5 ′ -ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5 ′ -ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5 ′ -hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4 + T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5 ′ -hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

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A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Cited by 169 publications

References 87 publications

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

CRISPR RNAs trigger innate immune responses in human cells

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