@article{5d491f3766114300ab8733d16228edc9,
title = "An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms",
abstract = "We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). 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 in vivo. The approach is fast, cheap, and scalable.",
author = "Oguz Kanca and Jonathan Zirin and Jorge Garcia-Marques and Knight, {Shannon Marie} and Donghui Yang-Zhou and Gabriel Amador and Hyunglok Chung and Zhongyuan Zuo and Liwen Ma and Yuchun He and Lin, {Wen Wen} and Ying Fang and Ming Ge and Shinya Yamamoto and Schulze, {Karen L.} and Yanhui Hu and Spradling, {Allan C.} and Mohr, {Stephanie E.} and Norbert Perrimon and Bellen, {Hugo J.}",
note = "Funding Information: The Drosophila Gene Disruption Project is supported by NIH NIGMS R01GM067858 to HJB. Confocal microscopy at the NRI is supported by the Neurovisualization Core of the IDDRC, funded by NIH U54HD083092. We thank the Harvard Medical School Image Data Management Core for support with the OMERO platform and the Harvard Medical School Division of Immunology{\textquoteright}s Flow Cytometry Facility for the use of their FACS equipment. We also thank Raghuvir Viswanatha, Justin Bosch, Denise Lanza and Jason Heaney for helpful discussions, Robert Levis for critical reading and editing of the manuscript and Tzumin Lee for fly stocks. Development of the tagged cell line resource at the Drosophila RNAi Screening Center (DRSC) was supported by NIH ORIP R24 OD019847 (PI: NP, Co-PI: A Simcox, Co-I: SEM). Additional relevant support for the DRSC includes NIH NIGMS R01 GM084947 and P41 GM132087 (PI: NP, Co-I: SEM). NP, ACS, and HB are investigators of Howard Hughes Medical Institute. Funding Information: NIH NIGMSR01GM067858 to HJB. Neurovisualization Core of the IDDRC, funded by NIH U54HD083092. Harvard Medical School Image Data Management Core for support with the OMEROHarvard Medical School Division of Immunology{\textquoteright}s Flow Cytometry Facility for the use of their FACS equipment. Drosophila RNAi Screening Center(DRSC) was supported by NIH ORIPR24 OD019847 (PI: NP, Co-PI: A Simcox, Co-I: SEM). Additional relevant support for the DRSC includes NIH NIGMSR01 GM084947 and P41 GM132087 (PI: NP, Co-I: SEM). NP, ACS, and HB are investigators of Howard Hughes Medical Institute. Publisher Copyright: {\textcopyright} 2019, eLife Sciences Publications Ltd. All rights reserved.",
year = "2019",
month = nov,
doi = "10.7554/eLife.51539",
language = "English (US)",
volume = "8",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications Ltd",
}