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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 476-489(2017) https://doi.org/10.1007/s11427-017-9029-9

Genome editing in Drosophila melanogaster: from basic genome engineering to the multipurpose CRISPR-Cas9 system

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  • ReceivedJan 20, 2017
  • AcceptedMar 5, 2017
  • PublishedMay 1, 2017

Abstract

Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods, ethyl methane sulfonate (EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level. Since the discovery of transposable elements and the availability of the complete fly genome, specific genome editing tools, such as P-elements, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have undergone rapid development. Currently, one of the leading and most effective contemporary tools is the CRISPR-cas9 system made popular because of its low cost, effectiveness, specificity and simplicity of use. This review briefly addresses the most commonly used mutagenesis and transgenesis tools in Drosophila, followed by an in-depth review of the multipurpose CRISPR-Cas9 system and its current applications.


Funded by

National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China(2015BAI09B03,2016YFE0113700)

National Natural Science Foundation of China(31371496,31571320)

National Basic Research Program(2013CB35102)


Acknowledgment

We thank members of the Ni lab for their critical comments on the manuscript. This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China (2015BAI09B03, 2016YFE0113700), the National Natural Science Foundation of China (31371496, 31571320), the National Basic Research Program (2013CB35102).


Interest statement

The author(s) declare that they have no conflict of interest.


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  • Figure 1

    Overview of genome engineering methods in Drosophila melanogaster throughout history. The Drosophila genome engineering methods allow researchers to modify the genome in a random (chemical mutagens, physical mutagens, transposable elements) or specific fashion (ZFNs, TALENs, CRISPR-Cas9 system). Chemical mutagens, e.g. EMS, provokes single nucleotide transitions leading to random point mutations, whereas physical mutagens, e.g. X-rays, create DNA DSBs introducing inversions, deletions or duplications of genomic fragments. Transposable elements, e.g. P-elements, on the other hand, are mobile DNA fragments that can disrupt the genome by insertion or excision of the fragment at almost random locations. The second group of genome editing methods provides a more specific way of modification by using a designed nuclease-DNA targeting complex to introduce a DSB at a target sequence of interest. ZFN and TALEN, both use the Fok I-nuclease which upon dimerization facilitates a DSB. The specificity is defined by the DNA binding domains of the complex, which for ZFNs and TALENs interacts with 3 and 1 bp per module, respectively. The CRISPR-Cas9 system uses the Cas9 nuclease instead of Fok I to target specific genomic loci. The Cas9 nuclease is directed to its target sequence by binding of a sgRNA, which has a 20 nucleotide DNA binding sequence at its 5′ end.

  • Figure 2

    HEI, FRT/FLP and Hobo strategies for deletional mutagenesis with transposable elements. HEI is a strategy that allows for the possibility of the transposition process to pair different ends (A). This event occurs favorably when two P-elements are in trans on sister chromatids, removing everything between the P-element regions. P-elements inserted using a FRT-FLP-cassette, can generate a deletion by FLP-recombinase in a same fashion as the HEI system (B). By strategically using the white+ marker gene, the mutant offspring can be easily selected when lacking the white+ phenotype. The hybrid P-element construct P[wHy] contains a Hobo deletion element flanked by two selection markers, namely white+ and yellow+ (C). When the Hobo element is duplicated to another genomic region via local hopping (1), the region in-between can be deleted by homologous recombination of the Hobo fragment (2). Mutants can be easily selected because they only show the white+ phenotype.

  • Figure 3

    Endogenous cellular DNA repair mechanisms. Generation of a DSB leads to the stimulation of the NHEJ and HDR endogenous cellular repair machineries. NHEJ does not need a template and often results in (1) altered nucleotides at the breakage area, (2) small deletions or (3) inserts. HDR repairs the DSB using a homologous template, which when provided in a vector construct, can be used to induce (4) insertions or deletions at the breakage site.

  • Table 1   Overview of the CRISPR-Cas9 system mediated NHEJ in

    NHEJ

    Cas9 and sgRNA information (format)

    Target gene

    Overall germline transmission rate, %*

    Reference

    hsp70-Cas9 (DNA)

    U6b-sgRNA (DNA)

    yellow

    0.25%–1.37%

    (Gratz et al., 2013)

    T7-Cas9 (mRNA)

    T7-sgRNA (RNA)

    yellowwhite

    0.0%–34.5%

    (Bassett et al., 2013)

    SP6-Cas9 (mRNA)

    T7-sgRNA (RNA)

    yellow

    8.10%–10.40%

    (Yu et al., 2013)

    nos-Cas9 (transgene)

    U6b-sgRNA (transgene)

    white

    3.4%–93.2%

    (Kondo and Ueda, 2013)

    nos-Cas9 (transgene)

    U6b-sgRNA (DNA)

    white

    12.4%–74.2%

    (Ren et al., 2013)

    vasa-Cas9 (transgene)

    U6b-sgRNA (DNA)

    rosy

    6.49%–15.11%

    (Gratz et al., 2014)

    vasa-Cas9 (transgene)

    U6-sgRNA (DNA)

    3×P3-mRFP

    3×P3-EGFP

    0%–100%**

    (Sebo et al., 2014)

    actin5C-Cas9 (transgene)

    vasa-Cas9 (transgene)

    nos-Cas9 (transgene)

    nos-Cas9:GFP (transgene)

    nosG4VP16>UAS-Cas9 (transgene)

    U6c-sgRNA (transgene)

    yellow

    ebony

    26.1%–100%

    (Port et al., 2014)

    U6b-sgRNA-hsp70Bb-Cas9 (DNA)***

    ebony

    13.0%

    (Gokcezade et al., 2014)

    *, The overall germline transmission rate is calculated as the number of mutant offspring divided by the total number of offspring from all crosses. **, The efficiency is the individual germline transmission rate instead of overall germline transmission rate. ***, Cas9 under the control of the hsp70Bb promoter and sgRNA under the control of U6b promoter are encoded on a single DNA plasmid.

  • Table 2   Overview of the CRISPR-Cas9 system mediated HDR in

    HDR

    Cas9 and sgRNA information (format)

    Donor

    Target gene

    Genomic modification

    Overall germline transmission rata, % (n)*

    Reference

    hsp70-Cas9 (DNA)

    U6b-sgRNA (DNA)

    ssODN donor

    yellow

    End-out

    0.04%**

    (Gratz et al., 2013)

    vasa-Cas9 (transgene)

    U6b-sgRNA (DNA)

    dsDNA donor

    DSH3PX1

    End-out

    1.98% (599/7657)***

    7.82% (45/2277)****

    (Gratz et al., 2014)

    Cas9 (mRNA)

    sgRNA (RNA)

    dsDNA donor

    CG4221

    loxP replacement

    4.3% (10/230)

    (Yu et al., 2014)

    CG5961

    Hind III replacement

    3.8% (2/52)

    Chameau

    eGFP tagging

    2.7% (8/296)

    CG4221

    Myc tagging

    10.4% (24/231)

    nos-Cas9 (transgene)

    U6:3-sgRNA-wls (transgene)

    ssODN

    wntless

    amino acid point mutation

    (Gly11Ala: GGC-GcC)

    28% (13/46)

    (Port et al., 2014)

    nos-Cas9 (transgene)

    U6b-sgRNA (DNA)

    dsDNA donor

    piwi

    End-out

    32.8% (446/1361)

    (Ren et al., 2014b)

    hp1a

    12% (98/822)

    *, The overall germline transmission rate is calculated as the number of mutant offspring divided by the total number of offspring from all crosses. **, The overall germline transmission rate of yellow mutation is 1.16% ((24+34)/(2336+2655)=1.16%), and the HDR transmission events only occurred in 2 founders of 61 G0 flies. Therefore, the estimated overall germline transmission rate of HDR is 0.04% (1.16%×3.28%=0.04%). ***, sgRNAS1+S2. ****, sgRNAS1+S3.

  • Table 3   Online resources for finding genome-wide sgRNAs

    Name

    Link

    Contributor

    Reference

    DRSC Find CRISPRs

    http://www.flyrnai.org/crispr2/

    Norbert Perrimon lab

    (Ren et al., 2013)

    E-CRISPR

    http://www.e-crisp.org/E-CRISP/

    Michael Boutros lab

    (Heigwer et al., 2014)

    CRISPR optimal target finder

    http://tools.flycrispr.molbio.wisc.edu/targetFinder/

    KateM. O’Connor-Giles lab

    (Gratz et al., 2014)

    FlyCas9

    http://shigen.nig.ac.jp/fly/nigfly/cas9/index.jsp

    Shu Kondo lab

    (Kondo and Ueda, 2013)

    CRISPR Design

    http://www.genome-engineering.org/crispr/?page_id=41

    Feng Zhang lab

    (Cong et al., 2013)

    (Hsu et al., 2013)

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