Functional Editing of Endogenous Genes Through Rapid Selection of Cell Pools (Rapid Generation of Endogenously Tagged Genes in Drosophila Ovarian Somatic Sheath Cells)

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2022 May 31

PMID 35639929

Authors

Qingcai Meng

Daniel Stoyko

Celine Marlin Andrews

Parthena Konstantinidou

Pavol Genzor

Timothy O

Alexandra R Elchert

Leif Benner

Sushil Sobti

Esther Y Katz

Astrid D Haase

Affiliations

Soon will be listed here.

Abstract

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.

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References

Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S . Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011; 9(1):72-4. DOI: 10.1038/nmeth.1778. View

Yamashiro H, Siomi M . PIWI-Interacting RNA in Drosophila: Biogenesis, Transposon Regulation, and Beyond. Chem Rev. 2017; 118(8):4404-4421. DOI: 10.1021/acs.chemrev.7b00393. View

Palmiter R, Brinster R . Transgenic mice. Cell. 1985; 41(2):343-5. DOI: 10.1016/s0092-8674(85)80004-0. View

Muerdter F, Guzzardo P, Gillis J, Luo Y, Yu Y, Chen C . A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila. Mol Cell. 2013; 50(5):736-48. PMC: 3724422. DOI: 10.1016/j.molcel.2013.04.006. View

Zhang X, Guo C, Chen Y, Shulha H, Schnetz M, LaFramboise T . Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nat Methods. 2008; 5(2):163-5. PMC: 2435063. DOI: 10.1038/nmeth1170. View

Perez A, Pritykin Y, Vidigal J, Chhangawala S, Zamparo L, Leslie C . GuideScan software for improved single and paired CRISPR guide RNA design. Nat Biotechnol. 2017; 35(4):347-349. PMC: 5607865. DOI: 10.1038/nbt.3804. View

Aravin A, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D, Snyder B . The small RNA profile during Drosophila melanogaster development. Dev Cell. 2003; 5(2):337-50. DOI: 10.1016/s1534-5807(03)00228-4. View

Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E . A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature. 2009; 461(7268):1296-9. DOI: 10.1038/nature08501. View

Ozata D, Gainetdinov I, Zoch A, OCarroll D, Zamore P . PIWI-interacting RNAs: small RNAs with big functions. Nat Rev Genet. 2018; 20(2):89-108. DOI: 10.1038/s41576-018-0073-3. View

10.

Vagin V, Sigova A, Li C, Seitz H, Gvozdev V, Zamore P . A distinct small RNA pathway silences selfish genetic elements in the germline. Science. 2006; 313(5785):320-4. DOI: 10.1126/science.1129333. View

11.

Kanca O, Bellen H, Schnorrer F . Gene Tagging Strategies To Assess Protein Expression, Localization, and Function in . Genetics. 2017; 207(2):389-412. PMC: 5629313. DOI: 10.1534/genetics.117.199968. View

12.

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

13.

Stein C, Genzor P, Mitra S, Elchert A, Ipsaro J, Benner L . Decoding the 5' nucleotide bias of PIWI-interacting RNAs. Nat Commun. 2019; 10(1):828. PMC: 6381166. DOI: 10.1038/s41467-019-08803-z. View

14.

Haase A, Fenoglio S, Muerdter F, Guzzardo P, Czech B, Pappin D . Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 2010; 24(22):2499-504. PMC: 2975926. DOI: 10.1101/gad.1968110. View

15.

Niki Y, Yamaguchi T, Mahowald A . Establishment of stable cell lines of Drosophila germ-line stem cells. Proc Natl Acad Sci U S A. 2006; 103(44):16325-30. PMC: 1637581. DOI: 10.1073/pnas.0607435103. View

16.

Sienski G, Donertas D, Brennecke J . Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression. Cell. 2012; 151(5):964-80. PMC: 3504300. DOI: 10.1016/j.cell.2012.10.040. View

17.

Jin Y, Tam O, Paniagua E, Hammell M . TEtranscripts: a package for including transposable elements in differential expression analysis of RNA-seq datasets. Bioinformatics. 2015; 31(22):3593-9. PMC: 4757950. DOI: 10.1093/bioinformatics/btv422. View

18.

Vandemoortele G, Eyckerman S, Gevaert K . Pick a Tag and Explore the Functions of Your Pet Protein. Trends Biotechnol. 2019; 37(10):1078-1090. DOI: 10.1016/j.tibtech.2019.03.016. View

19.

Hafner M, Renwick N, Farazi T, Mihailovic A, Pena J, Tuschl T . Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods. 2012; 58(2):164-70. PMC: 3508525. DOI: 10.1016/j.ymeth.2012.07.030. View

20.

Knott G, Doudna J . CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361(6405):866-869. PMC: 6455913. DOI: 10.1126/science.aat5011. View