Abstract
Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR (2019) Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576(7785):149–157. https://doi.org/10.1038/s41586-019-1711-4
Scholefield J, Harrison PT (2021) Prime editing – an update on the field. Gene Ther 28(7):396–401. https://doi.org/10.1038/s41434-021-00263-9
Kim DY, Moon SB, Ko JH, Kim YS, Kim D (2020) Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Res 48(18):10576–10589. https://doi.org/10.1093/nar/gkaa764
Jin S, Lin Q, Luo Y, Zhu Z, Liu G, Li Y, Chen K, Qiu JL, Gao C (2021) Genome-wide specificity of prime editors in plants. Nat Biotechnol 39(10):1292–1299. https://doi.org/10.1038/s41587-021-00891-x
Rees HA, Liu DR (2018) Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19(12):770–788. https://doi.org/10.1038/s41576-018-0059-1
Choi J, Chen W, Suiter CC, Lee C, Chardon FM, Yang W, Leith A, Daza RM, Martin B, Shendure J (2021) Precise genomic deletions using paired prime editing. bioRxiv 40(2):218–226. https://doi.org/10.1101/2020.12.30.424891
Hsu JY, Grunewald J, Szalay R, Shih J, Anzalone AV, Lam KC, Shen MW, Petri K, Liu DR, Joung JK, Pinello L (2021) PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun 12(1):1034. https://doi.org/10.1038/s41467-021-21337-7
Bosch JA, Birchak G, Perrimon N (2021) Precise genome engineering in Drosophila using prime editing. Proc Natl Acad Sci U S A 118(1):e2021996118. https://doi.org/10.1073/pnas.2021996118
Kim HK, Yu G, Park J, Min S, Lee S, Yoon S, Kim HH (2021) Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol 39(2):198–206. https://doi.org/10.1038/s41587-020-0677-y
Lin Q, Jin S, Zong Y, Yu H, Zhu Z, Liu G, Kou L, Wang Y, Qiu JL, Li J, Gao C (2021) High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol 39(8):923–927. https://doi.org/10.1038/s41587-021-00868-w
Hurd TR, Liang FX, Lehmann R (2015) Curly encodes dual oxidase, which acts with Heme peroxidase curly Su to shape the adult drosophila wing. PLoS Genet 11(11):e1005625. https://doi.org/10.1371/journal.pgen.1005625
Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in drosophila. Proc Natl Acad Sci U S A 111(29):E2967–E2976. https://doi.org/10.1073/pnas.1405500111
Port F, Bullock SL (2016) Augmenting CRISPR applications in drosophila with tRNA-flanked sgRNAs. Nat Methods 13(10):852–854. https://doi.org/10.1038/nmeth.3972
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3--new capabilities and interfaces. Nucleic Acids Res 40(15):e115. https://doi.org/10.1093/nar/gks596
Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, Cole MA, Liu DR, Joung JK, Bauer DE, Pinello L (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37(3):224–226. https://doi.org/10.1038/s41587-019-0032-3
Kweon J, Yoon JK, Jang AH, Shin HR, See JE, Jang G, Kim JI, Kim Y (2021) Engineered prime editors with PAM flexibility. Mol Ther 29(6):2001–2007. https://doi.org/10.1016/j.ymthe.2021.02.022
Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N (2015) Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal 8(393):rs9. https://doi.org/10.1126/scisignal.aab3729
Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196(4):961–971. https://doi.org/10.1534/genetics.113.160713
Anderson MV, Haldrup J, Thomsen EA, Wolff JH, Mikkelsen JG (2021) pegIT – a web-based design tool for prime editing. Nucleic Acids Res 49(W1):W505–W509. https://doi.org/10.1093/nar/gkab427
Chow RD, Chen JS, Shen J, Chen S (2021) A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng 5(2):190–194. https://doi.org/10.1038/s41551-020-00622-8
Siegner SM, Karasu ME, Schroder MS, Kontarakis Z, Corn JE (2021) PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants. BMC Bioinformatics 22(1):101. https://doi.org/10.1186/s12859-021-04034-6
Hwang GH, Jeong YK, Habib O, Hong SA, Lim K, Kim JS, Bae S (2021) PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res 49(W1):W499–W504. https://doi.org/10.1093/nar/gkab319
Adikusuma F, Lushington C, Arudkumar J, Godahewa G, Chey YCJ, Gierus L, Pilitz S, Reti D, Wilson LOW, Bauer DC, Thomas PQ (2021) Optimized nickase- and nuclease-based prime editing in human and mouse cells. Nucleic Acids Res 49(18):10785–10795
Morris JA, Rahman JA, Guo X, Sanjana NE (2020) Automated design of CRISPR prime editors for thousands of human pathogenic variants. bioRxiv. https://doi.org/10.1101/2020.05.07.083444
Standage-Beier K, Tekel SJ, Brafman DA, Wang X (2021) Prime editing guide RNA design automation using PINE-CONE. ACS Synth Biol 10(2):422–427. https://doi.org/10.1021/acssynbio.0c00445
Li Y, Chen J, Tsai SQ, Cheng Y (2021) Easy-prime: a machine learning-based prime editor design tool. Genome Biol 22(1):235. https://doi.org/10.1186/s13059-021-02458-0
Acknowledgments
The authors thank Gabriel Birchak for discussions and S2R+ work, Rich Binari for general lab assistance and help with fly, the TRiP and Drosophila RNAi Screening Center for help generating transgenic flies, Ram Viswanatha for general discussions, Gillian Millburn for discussions on pegRNA transgene nomenclature, Cathryn Murphy for general lab assistance, Cooper Cavers for help isolating transgenic flies, Jorden Rabasco for help with molecular cloning, Andrew Anzalone for advice with synthetic pegRNAs, and Ben Ewen-Campen, Jonathan Zirin, and Thai LaGraff for cloning help. J.A.B. was supported by the Damon Runyon Foundation (DRG-2258-16) and a “Training Grant in Genetics” T32 Ruth Kirschstein-National Research Service Award institutional research training grant funded through the NIH/National Institute of General Medical Sciences (T32GM007748). This work was also supported by NIH Grant P41GM132087. N.P. is an Investigator of the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bosch, J.A., Perrimon, N. (2022). Prime Editing for Precise Genome Engineering in Drosophila. In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 2540. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2541-5_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2541-5_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2540-8
Online ISBN: 978-1-0716-2541-5
eBook Packages: Springer Protocols