in vivo fly CRISPR

R. Viswanatha, M. Zaffagni, J. Zirin, N. Perrimon, and S. Kadener. Submitted. “CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells.” BioRxiv.Abstract
Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.
J. A. Bosch, G. Birchak, and N. Perrimon. 2021. “Precise genome engineering in Drosophila using prime editing.” Proc Natl Acad Sci U S A, 118.Abstract
Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.
Image of an anesthetized male Drosophila fruit fly

DRSC/TRiP presentations from June 2020 Boston Area Drosophila Meeting

June 12, 2020
Did you miss the presentations from Claire Hu and Jonathan Zirin at the June 2020 Boston Area Drosophila Meeting? No problem! The slides can be accessed from this post. Click the title above to view the whole post, then scroll down to access the PDFs. These presentations describe what's new and next in bioinformatics and in vivo technologies at the DRSC/TRiP. Feel free to reach out with questions. Interested in the BAD meeting? Info about the meeting can be found here. Read more about DRSC/TRiP presentations from June 2020 Boston Area Drosophila Meeting
from Figure 1 in Ewen-Campen et al. in Dev Cell

Transgenic Fly Stocks for Double Knockout of Paralog Pairs

May 18, 2020

Paralogs can be defined as related genes within a genome that are thought to arise from gene duplication events. Because paralogous proteins share amino acid identity, they can have redundant functions. But the picture is not necessarily so straightforward. Indeed, there are examples in which paralogous genes have distinct functions in some tissues, and overlapping functions in others.

The DRSC/TRiP is engaged in a project in collaboration with the Perrimon and Bellen labs to generate resources useful for the study of paralogous genes in Drosophila.

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Read more about Transgenic Fly Stocks for Double Knockout of Paralog Pairs
Jonathan Zirin, Yanhui Hu, Luping Liu, Donghui Yang-Zhou, Ryan Colbeth, Dong Yan, Ben Ewen-Campen, Rong Tao, Eric Vogt, Sara VanNest, Cooper Cavers, Christians Villalta, Aram Comjean, Jin Sun, Xia Wang, Yu Jia, Ruibao Zhu, Ping Peng, Jinchao Yu, Da Shen, Yuhao Qiu, Limmond Ayisi, Henna Ragoowansi, Ethan Fenton, Senait Efrem, Annette Parks, Kuniaki Saito, Shu Kondo, Liz Perkins, Stephanie E Mohr, Jianquan Ni, and Norbert Perrimon. 2020. “Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies.” Genetics.Abstract
The Transgenic RNAi Project (TRiP), a functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes. Cutting is triggered by co-expression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.
Oguz Kanca, Jonathan Zirin, Jorge Garcia-Marques, Shannon Marie Knight, Donghui Yang-Zhou, Gabriel Amador, Hyunglok Chung, Zhongyuan Zuo, Liwen Ma, Yuchun He, Wen-Wen Lin, Ying Fang, Ming Ge, Shinya Yamamoto, Karen L Schulze, Yanhui Hu, Allan C Spradling, Stephanie E Mohr, Norbert Perrimon, and Hugo J Bellen. 2019. “An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.” Elife, 8.Abstract
We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an - transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely . The approach is fast, cheap, and scalable.
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC-Biomedical Technology Research Resource

October 21, 2019

We are pleased to announce that we have been funded by NIH NIGMS to form the Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR). The P41-funded DRSC-BTRR (N. Perrimon, PI; S. Mohr, Co-I) builds upon and extends past goals of the Drosophila RNAi Screening Center.

As the DRSC-BTRR, we are working together with collaborators whose 'driving biomedical projects' inform development of new technologies at the DRSC. At the same time, we continue to support Drosophila cell-based RNAi and CRIPSR knockout screens and related...

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Figure 1 from the Escobedo et al. micropublication

Micropublication relevant to TRiP fly stocks

April 9, 2019

Users of TRiP RNAi and sgRNA fly stocks take note: the Weake lab at Purdue University brought to our attention that some TRiP fly stocks carry a mutant allele of seveneless. Jonathan Zirin worked with Spencer Escobedo and Vikki Weake, as well as with folks at the Bloomington Drosophila Stock Center, to quickly identify the source, sequence the mutant allele, and pubilsh a micropublication so we can get the details to the community. Bottom line, as stated in the micropublication, "The presence of the sev[21]  mutation will not generally affect the use of these stocks, as the X...

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flySAM

Missed us at ADRC 2018? View our workshop slides!

April 19, 2018
Thank you to all those who attended our workshop at last week's Annual Drosophila Research Conference in Philadelphia, PA, USA. It was great to talk fly stocks, cell screens, and bioinformatics with the community. We are here to help and look forward to continued feedback on the resources we are building to empower your research. PDFs of our workshop presentations are attached to this news item. The slides will help you learn more about our in vivo resources for CRISPR, new pooled cell-based CRISPR screen technology, and bioinformatics resources at our facility.  Feel free to contact... Read more about Missed us at ADRC 2018? View our workshop slides!
2018 Apr 13

DRSC & TRiP Workshop at ADRC

1:45pm to 3:45pm

Location: 

Philadelphia, PA, USA
The DRSC & TRiP will be hosting a workshop at the Annual Drosophila Research Conference in Philadelphia, PA. The workshop is scheduled for Friday, April 13th from 1:45 to 3:45 PM. Come hear from DRSC & TRiP leaders Norbert Perrimon, Jonathan Zirin (organizer), Claire Yanhui Hu, and Stephanie Mohr. At the workshop, you will learn about new opportunities for community nomination and experiments using CRISPR knockout and activation, as well as learn what's new and popular among our online software and database tools. There will be something for everyone -- we will provide information... Read more about DRSC & TRiP Workshop at ADRC
Ben Ewen-Campen, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 10/9/2017. “Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR.” Dev Cell, 43, 1, Pp. 6-9.Abstract
Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.

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