Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles

Michlits, Georg; Jude, Julian; Hinterndorfer, Matthias; Almeida, Melanie de; Vainorius, Gintautas; Hubmann, Maria; Neumann, Tobias; Schleiffer, Alexander; Burkard, Thomas R; Fellner, Michaela; Gijsbertsen, Max; Traunbauer, Anna K.; Zuber, Johannes; Elling, Ulrich

doi:10.1038/s41592-020-0850-8

Cited by 97 publications

(104 citation statements)

References 49 publications

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“…Obviously, the analysis of such experiments would be challenging in the F 0 generation but achieving dominant editing of specific in-frame variants heavily increases the chance of passing the exact genotype of interest to the F 1 generation. Furthermore, to deal with the possible NMD-induced transcriptional adaptation and compensation mentioned above, it was recently argued that the use of gRNAs selecting for in-frame mutations in domains essential for protein function could be another approach to produce loss-of-function alleles 37 . In this scope, InDelphi could similarly be employed to identify gRNAs with high predicted in-frame frequencies (our lowest-in-class gRNAs).…”

Section: Discussionmentioning

confidence: 99%

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

Naert

Tulkens

Edwards

et al. 2020

Sci Rep

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CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F 0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F 0 generation. Over the last couple of years, CRISPR/Cas9 has revolutionized reverse genetic studies in non-mammalian vertebrate model organisms 1-3 , and has further empowered the use of Xenopus and zebrafish as model organisms for studying development and human disease 4-6. In particular, F 0 CRISPR/Cas9-mediated gene disruption in non-mammalian vertebrates has emerged as a cost-effective method to rapidly assign causality to genetic variants in candidate disease genes identified from human patient exome sequencing 7-11. This can assist clinical geneticists in providing timely genetic diagnosis and counseling to patients and affected families, thereby favoring societal and economic impact of findings. CRISPR/Cas9 mediated F 0 mosaic mutant embryos are also increasingly employed as an alternative to antisense morpholino oligomers (MOs) 12,13 to investigate gene function and genetic interactions in developing embryos 14 , thus expanding the toolbox for cell and developmental biologists. An important consideration in CRISPR/Cas9 mutational studies is identifying gRNAs that produce a high frequency of loss-of-function mutations in the appropriate coding exons and hence generate highly penetrant specific F 0 phenotypes 15. During gRNA design, considerations include the possibilities of reading frame preservation

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Section: Discussionmentioning

confidence: 99%

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

Naert

Tulkens

Edwards

et al. 2020

Sci Rep

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“…Cloning of the Gordon et al SARS-CoV-2 Human Interactome CRISPR Library sgRNA sequences (10 per gene) targeting 332 host factor proteins recently shown to interact with SARS-CoV-2 proteins (Gordon et al, 2020) (Table S1A) were designed using a combination of the Vienna Bioactivity CRISPR score (Michlits et al, 2020) and the Broad Institute sgRNA Designer tool (Doench et al, 2016). In addition, a subset of sgRNAs were hand-picked from the Wang-Sabatini genome-wide libraries (Wang et al, 2015).…”

Section: Materials and Methods (Supplementarymentioning

confidence: 99%

“…In addition, a subset of sgRNAs were hand-picked from the Wang-Sabatini genome-wide libraries (Wang et al, 2015). We also included 314 safe-targeting sgRNAs (Morgens et al, 2017) and 310 sgRNAs targeting essential genes (Hart et al, 2017;Michlits et al, 2020) for a total of 3,944 sgRNAs. We refer to this library as the 'Gordon et al SARS-CoV-2 Human…”

Section: Materials and Methods (Supplementarymentioning

confidence: 99%

“…We constructed a lentiviral transfer plasmid library encoding 3,944 sgRNAs designed to disrupt all 332 human genes identified in the abovementioned SARS-CoV-2 interactome ( Figure 1A). The library includes 10 sgRNAs targeting each gene, 314 safe-targeting sgRNAs (which cut the genome at non-genic regions (Morgens et al, 2017)), and 310 sgRNAs targeting genes essential for cell survival or proliferation (Hart et al, 2017;Michlits et al, 2020). We used this library to perform a pooled CRISPR-Cas9 gene disruption screen in Huh-7.5 hepatoma cells stably expressing Cas9 (Huh-7.5-Cas9) ( Figure 1B).…”

Section: A Focused Crispr-cas9 Screen Functionally Validates Host Promentioning

confidence: 99%

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Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

Hoffmann

Schneider

Sánchez‐Rivera

et al. 2020

Preprint

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SUMMARYThe ongoing SARS-CoV-2 pandemic has devastated the global economy and claimed nearly one million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen four related coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and SARS-CoV-2) at two physiologically relevant temperatures (33 °C and 37 °C), allowing us to probe this interactome at a much higher resolution relative to genome scale studies. This approach yielded several new insights, including unexpected virus and temperature specific differences in Rab GTPase requirements and GPI anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating COVID-19, and help prepare for future coronavirus outbreaks.HIGHLIGHTSFocused CRISPR screens targeting host factors in the SARS-CoV-2 interactome were performed for SARS-CoV-2, HCoV-229E, HCoV-NL63, and HCoV-OC43 coronaviruses.Focused interactome CRISPR screens achieve higher resolution compared to genome-wide screens, leading to the identification of critical factors missed by the latter.Parallel CRISPR screens against multiple coronaviruses uncover host factors and pathways with pan-coronavirus and virus-specific functional roles.The number of host proteins that interact with a viral bait protein is not proportional to the number of functional interactors.Novel SARS-CoV-2 host factors are expressed in relevant cell types in the human airway.

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“…Says Hyongbum Henry Kim, a researcher at Yonsei University College of Medicine in Seoul, "I am excited to hear that Nobel Prize is given to the field of my research. " As the field basks in Nobel glory and gallops on, the gRNA family will continue to grow and diversify, and so will ways to assess gRNAs [1][2][3][4][5] . There's more to gRNA choice and use than the ' Add to Cart' click on an Addgene order.…”

mentioning

confidence: 99%

Guide RNAs: it’s good to be choosy

Marx

2020

Nat Methods

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Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles

Cited by 97 publications

References 49 publications

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

Guide RNAs: it’s good to be choosy

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