Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

Replogle, Joseph M.; Bonnar, Jessica L.; Pogson, Angela N; Liem, Christina R; Maier, Nolan K; Ding, Yufang; Russell, Baylee J.; Xing-ren, Wang; Leng, Kun; Guna, Alina; Norman, Thomas M.; Pak, Ryan A.; Ramos, Daniel M.; Ward, Michael E.; Gilbert, Luke A.; Kampmann, Martin; Weissman, Jonathan S.; Jost, Marco

doi:10.1101/2022.07.13.499814

Cited by 9 publications

(13 citation statements)

References 75 publications

(91 reference statements)

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“…CRISPRi is highly efficient at suppressing gene expression of selected targets without introducing double-strand breaks, with minimal offtarget effects. On-target activity can be maximized by using two single-guide RNAs (sgRNAs) per target, expressed from one lentiviral vector (35)(36)(37).…”

Section: Functional Genomics Of Coronavirus Host Factors With a Singl...mentioning

confidence: 99%

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Sunshine

Puschnik

Replogle

et al. 2022

Preprint

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Numerous host factors of SARS-CoV-2 have been identified by screening approaches, but delineating their molecular roles during infection and whether they can be targeted for antiviral intervention remains a challenge. Here we use Perturb-seq, a single-cell CRISPR screening approach, to investigate how CRISPR interference of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our data reveal two classes of host factors with pronounced phenotypes: factors required for the response to interferon and factors required for entry or early infection. Among the latter, we have characterized the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides high-throughput functional validation of host factors of SARS-CoV-2 and describes their roles during viral infection in both infected and bystander cells.

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Section: Functional Genomics Of Coronavirus Host Factors With a Singl...mentioning

confidence: 99%

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Sunshine

Puschnik

Replogle

et al. 2022

Preprint

Self Cite

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“…The optimizations developed here can be used to clone even more compact libraries, such as multi-guide Cas9 and Cas12a sgRNA constructs for CRISPRi and CRISPRa perturbations (51, 52). With these multi-guide libraries, it is conceivable to have a guide library containing a single element per gene.…”

Section: Discussionmentioning

confidence: 99%

Optimized CRISPR guide RNA library cloning reduces skew and enables more compact genetic screens

Heo

Enriquez

Federman

et al. 2022

Preprint

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The development of CRISPR genetic screening tools has improved functional genomics, as these tools enable precise genomic editing, provide broad access to genomic regions beyond protein-coding genes, and have fewer off-target effects than other functional genomics modalities, allowing for novel applications with smaller library sizes. Pooled functional genomics screens require high cellular coverage per perturbation to accurately quantify phenotypes and average out phenotype-independent variability across the population. While more compact libraries have decreased the number of cells needed for a given screen, the cell coverage required still poses technical hurdles to screen in more challenging systems, such as iPSC-derived and primary cells. A major factor that influences cell coverage is screening library uniformity, as larger variation in individual guide RNA abundance requires higher cell coverage to reliably measure low-abundance guides. In this work, we have systematically optimized guide RNA cloning procedures to decrease bias. We implement these protocols to demonstrate that CRISPRi screens using 10-fold fewer cells than the current standard provide equivalent statistically significant hit-calling results to screens run at higher coverage, opening the possibility of conducting genome-wide and other large-scale CRISPR screens in technically challenging models.

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“…CRISPRi screens were performed as described previously 65,82 . Briefly, HEI-193 cells stably expressing CRISPRi components (dCas9-KRAB) were transduced with lentivirus supernatant containing the third generation dual sgRNA CRISPRi library, which targets 20528 genes and 1025 sgNTC 82 . Screens were performed in triplicate cultures with coverage of at least 500x cells per construct.…”

Section: Methodsmentioning

confidence: 99%

“…Genomic DNA was harvested using the NucleoSpin Blood L Kit (Machery-Nagel) for each cell population, and sgRNA cassettes were amplified using 22 cycles of PCR using NEBNext Ultra II Q5 PCR MasterMix (New England Biolabs). Sequencing was performed on a NovaSeq 6000 (Illumina) using custom sequencing primers 82 .…”

Section: Crispr Interference Genome Wide Screeningmentioning

confidence: 99%

Epigenetic reprogramming shapes the cellular landscape of schwannoma

Liu

Casey-Clyde

Cho

et al. 2022

Preprint

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Cell state evolution underlies tumor development and response to therapy, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation. Schwannomas can oscillate in size for many years after radiotherapy, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

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Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

Cited by 9 publications

References 75 publications

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Optimized CRISPR guide RNA library cloning reduces skew and enables more compact genetic screens

Epigenetic reprogramming shapes the cellular landscape of schwannoma

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