1. Genetics and Genomics
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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

  1. David Li-Kroeger
  2. Oguz Kanca
  3. Pei-Tseng Lee
  4. Sierra Cowan
  5. Michael T Lee
  6. Manish Jaiswal
  7. Jose Luis Salazar
  8. Yuchun He
  9. Zhongyuan Zuo
  10. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Rice University Houston, United States
  3. Jan and Dan Duncan Neurological Research Institute, United States
Tools and Resources
Cite this article as: eLife 2018;7:e38709 doi: 10.7554/eLife.38709
6 figures, 3 tables, 1 data set and 2 additional files

Figures

Figure 1 with 2 supplements
Double Header optimizes RMCE outcome of MiMICs.

(A) Schematics of the Double Header construct and RMCE outcomes. Double Header constructs contain a Splice Acceptor (SA)- super folder GFP-FlAsH-StrepII-TEV-3xFlag (EGFP) – Splice Donor (SD) in one orientation and a SA-T2A-GAL4-polyA in the other orientation. Insertion in the GFP orientation results in GFP protein trap whereas insertion in the T2A-GAL4 orientation results in T2A-GAL4 gene trap. (B) Double Header injection statistics.

https://doi.org/10.7554/eLife.38709.003
Figure 1—figure supplement 1
Injection data for Double Header.

(A) The crossing scheme to generate Double Header RMCE events through embryo injection. (B) PCR determination of the Double Header orientation. ? indicates the number of lines where the PCR pattern was ambiguous. Yellow MiMICs: Double Header insertions in both orientations, Green MiMICs: only GFP protein trap orientation, Blue MiMICs: only T2A-GAL4 gene trap orientation.

https://doi.org/10.7554/eLife.38709.004
Figure 1—figure supplement 2
PCR strategy to identify Double Header orientation.

Primers are indicated on the Double Header construct that inserted in an intronic MiMIC. For each Double Header insertion four single fly PCR reactions were set with MiMIC inwards primers that bind outside the attP sites and are directed inwards (MiMIC_5’_for, MiMIC_3’_rev) and GFP and T2A-GAL4 specific outwards primers (DH_GFP_rev, DH_T2A-for). A correct insertion results in amplicons in two out of four PCRs.

https://doi.org/10.7554/eLife.38709.005
Figure 2 with 1 supplement
Double Header integration through crosses facilitates RMCE.

(A) Schematics of the Double Header transgene mobilization in vivo. Double Header transgenes contain loxP sites that can be used to mobilize the RMCE cassette in vivo, without the need for injection. (B) Double header crossing statistics.

https://doi.org/10.7554/eLife.38709.006
Figure 2—figure supplement 1
Crossing scheme for Double Header and data of integration.

(A) Crossing scheme for mobilizing Double Header RMCE cassette and selecting RMCE events. (B) PCR determination of the Double Header orientation. ? indicates the number of lines where PCR pattern was ambiguous. Yellow MiMICs: Double Header insertions in both orientations, Green MiMICs: only GFP protein trap orientation, Blue MiMICs: only T2A-GAL4 gene trap orientation.

https://doi.org/10.7554/eLife.38709.007
Figure 3 with 1 supplement
Examples of gene expression patterns obtained by Double Header.

Each MiMIC, MI01487, MI05208, MI06794, MI06872, MI08614, MI11741 and MI15073, was converted to either T2A-GAL4 protein traps or GFP protein traps by Double Header insertion. The expression in the larval CNS is shown with either T2A-GAL4 > UAS-mCD8::GFP or GFP-tag (GFP and mCD8::GFP, green). The affected genes are labelled above. Scale bar: 50 µm.

https://doi.org/10.7554/eLife.38709.008
Figure 3—figure supplement 1
Examples of gene expression patterns obtained by Double Header insertions in MiMICs in adult brain.

Each MiMIC MI01487, MI05208, MI06794, MI06872, MI08614, MI11741 and MI15073, was converted to either a T2A-GAL4 gene trap or GFP protein trap by Double Header insertion. The expression in the adult brain is shown with either T2A-GAL4 > UAS-mCD8::GFP or GFP-tag (GFP and mCD8::GFP, green). The affected genes are labelled above. Arrow: Ellipsoid body (MI05208-5HT2B). Scale bar: 50 µm.

https://doi.org/10.7554/eLife.38709.009
Examples of cellular expression patterns and subcellular localization of tagged proteins in egg chambers at stage 9 and 10.

Double header GFP protein traps of MIMIC lines shown in Figure 3 were dissected and ovaries were stained with anti-GFP antibody (green) and DAPI (red). Arrowheads indicate features that are referred to in the text; border cells for kibra; nurse cells, follicle cels and oocytes for 5HT2B; GFP is broadly expressed and distributed for Lgr4; note the apical enrichment in follicle cells in CG34383; nuclear and cytoplasmic staining in nurse cells and follicle cells are observed in Dgk; centripedal cells cytoplasm is mostly labeled in CG12206; broad expression and localization with pole cell enrichment in CG9132. Scale bar: 50 µm.

https://doi.org/10.7554/eLife.38709.010
Figure 5 with 2 supplements
Schematic of a two-step system for scarless gene editing.

(A) In step 1, a cassette containing a dominant marker flanked by nucleotides GG and CC replaces an endogenous locus via Homology Directed Repair (HDR) following double strand breaks produced by Cas9 cleavage (marked by red arrowheads). The removal of the intervening sequence between the Cas9 cut sites alters the sgRNA target sequences (underlined) preventing cleavage of the donor construct or the modified DNA. Screening for the dominant marker facilitates identification of CRISPR gene editing events while the flanking nucleotides GG (boxed inset) and CC create novel Cas9 target sites, allowing subsequent excision. (B) In step two the insert is removed and replaced with any DNA via a second round of HDR with new sgRNA sequences, facilitating the scarless insertion of any DNA sequence desirable.

https://doi.org/10.7554/eLife.38709.011
Figure 5—figure supplement 1
Template vectors for cloning yellow expression constructs.

(A) Vectors for ΦC31-mediated site-specific integration with yellow wing and body expression constructs. (B) yellow wing and body expression constructs compatible with Golden Gate cloning for SIC insertion via HDR. (C) Template donors compatible with Golden Gate cloning for yellow cassette swapping via HDR.

https://doi.org/10.7554/eLife.38709.012
Figure 5—figure supplement 2
Mapping cis-regulatory modules for the yellow gene.

(A) Structure of the yellow dominant marker showing previously characterized enhancers for wing and body (Geyer and Corces, 1987). (B) regions tested for yellow expression (black lines) when fused to a minimal promoter and the yellow gene. The table at the bottom right indicates the presence/absence of dark pigmentation in either the body or the wing for each cis-regulatory module (CRM) tested.

https://doi.org/10.7554/eLife.38709.013
ywing2+ cassette swapping facilitates structure-function analyses.

(A) Schematic of the Nmnat::GFP::Nmnat donor construct for replacing the inserted ywing2+ SIC at the Nmnat locus. (B) Nmnat::GFP::Nmnat variants used in the structure function experiment. Red * denotes approximate location of altered sequence(s). (C) Images of adult brains of Nmnat::GFP::Nmnat wt (Top left) Nmnat::GFP::NmnatW129G (Bottom left) Nmnat::GFP::NmnatΔ251…257 (Top right) and Nmnat:GFP:NmnatC344S, C345S (bottom right).

https://doi.org/10.7554/eLife.38709.016

Tables

Table 1
Summary statistics for cassette knock-in experiments
https://doi.org/10.7554/eLife.38709.014
ConstructGenotype injected:No. independent positive lines obtainedLethalityRescue of lethality/failure to complement
ywing2+ ΔNmnaty1 M{nos-Cas9.P}ZH-2A w*6lethalGenomic Fragment (Zhai et al., 2006)/NmnatΔ4790–1
ywing2+ ΔStub1y1 M{nos-Cas9.P}ZH-2A w*4Viable/Fertile*ND
ywing2+ ΔUbqnyw iso#6; +/+; attP2(y-){nos-Cas9}2lethalFails to complement Ubqn1
ywing2+ ΔItp-r83Ay1 M{nos-Cas9.P}ZH-2A w*1lethalND
ywing2+ ΔCG18769y1 M{nos-Cas9.P}ZH-2A w*2lethalND
ywing2+ ΔCG13390y1 M{nos-Cas9.P}ZH-2A w*2lethalRescued by Genomic Fragment (this study)
ywing2+ ΔMed27yw iso#6; +/+; attP2(y-){nos-Cas9}1lethalRescued by Genomic Fragment (this study)
ywing2+ ΔCG11679yw iso#6; +/+; attP2(y-){nos-Cas9}2lethalRescued by genomic duplication BSC Dp(1:3) 304
Ywing2+ Δrhoy1 M{nos-Cas9.P}ZH-2A w*0NANA
ywing2+ Δamxyw iso#6; +/+; attP2(y-){nos-Cas9}2Female sterileRescued by Genomic Fragment
  1. *two of four lines

Table 2
Summary statistics for cassette swapping experiments
https://doi.org/10.7554/eLife.38709.015
ConstructInjected genotype:No. embryos injectedNo. fertile adultsNo vials with y- flies% of y- flies confirmed positive
Nmnat:GFP:Nmnat wt #1y1 M{nos-Cas9.P}ZH-2A w*;+; ywing2+ ΔNmnat/TM6B514746%
Nmnat:GFP:Nmnat wt #2y1 M{nos-Cas9.P}ZH-2A w*;+; ywing2+ ΔNmnat/TM6B60716521%
Nmnat:GFP:NmnatW129G #1y1 M{nos-Cas9.P}ZH-2A w*;+; ywing2+ ΔNmnat/TM6B6530--
Nmnat:GFP:NmnatW129G #2y1 M{nos-Cas9.P}ZH-2A w*;3 KB NMNAT GRC; ywing2+ ΔNmnat41831355%
Nmnat:GFP:NmnatΔ251…257y1 M{nos-Cas9.P}ZH-2A w*;3 KB NMNAT GRC; ywing2+ ΔNmnat496291124%
Nmnat:GFP:NmnatC344S, C345Sy1 M{nos-Cas9.P}ZH-2A w*;3 KB NMNAT GRC; ywing2+ ΔNmnat386301214%
Stub1:GFPy1 M{nos-Cas9.P}ZH-2A w*; ywing2+ ΔStub123562266%
CG11679:Flagywing2+ ΔCG11679/FM7 Kgal4,UAS GFP;+/+; attP2(y-){nos-Cas997612*333%
Med27:flagy1 M{nos-Cas9.P}ZH-2A w*;+; ywing2+ ΔMed2783317329%
Amx:GFPywing2+ Δamx;+/+; attP2(y-){nos-Cas9648340-
  1. *excluding FM7 homozygotes and hemizygotes

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(Drosophila
melanogaster)
Double Header Jump
Starter phase 0 on
chromosome II
This studyFly strain containing
DH flanked by LoxP
sites
Genetic
reagent
(D. melanogaster)
Double Header Jump
Starter phase one
on chromosome II
This studyFly strain containing
DH flanked by LoxP
sites
Genetic
reagent
(D. melanogaster)
Double Header Jump
Starter phase two on
chromosome II
This studyFly strain containing
DH flanked by LoxP
sites
Genetic
reagent
(D. melanogaster)
Double Header Jump
Starter phase 0 on
chromosome III
This studyFly strain containing
DH flanked by LoxP sites
Genetic
reagent
(D. melanogaster)
Double Header Jump
Starter phase one on
chromosome III
This studyFly strain containing
DH flanked by LoxP
sites
Genetic
reagent
(D. melanogaster)
Double Header Jump
Starter phase two on
chromosome III
This studyFly strain containing
DH flanked by LoxP sites
Genetic
reagent
(D. melanogaster)
MI01487 (kibra)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0040175;
RRID:BDSC_40175
Genetic
reagent
(D. melanogaster)
MI05208 [5-HT2B
(5-hydroxytryptamine
receptor 2B)]
Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0042994;
RRID:BDSC_42994
Genetic
reagent
(D. melanogaster)
MI06794 [Lgr4(Leucine-rich
repeat-containing G
protein-coupled receptor 4)]
Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0042179:
RRID:BDSC_42179
Genetic
reagent
(D. melanogaster)
MI06872 (CG34383)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0041111;
RRID:BDSC_41111
Genetic
reagent
(D. melanogaster)
MI08614 [Dgk
(Diacyl glycerol kinase)]
Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0044991;
RRID:BDSC_44991
Genetic
reagent
(D. melanogaster)
MI11741 (CG12206)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0056687;
RRID:BDSC_56687
Genetic
reagent
(D. melanogaster)
MI15073 (CG9132)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0059739;
RRID:BDSC_59739
Genetic
reagent
(D. melanogaster)
MI02926 (Pits)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0036165;
RRID:BDSC_36165
Genetic
reagent
(D. melanogaster)
MI00805 (CG6966)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0034113;
RRID:BDSC_34113
Genetic
reagent
(D. melanogaster)
MI12643 (fz)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0058645;
RRID:BDSC_58645
Genetic
reagent
(D. melanogaster)
MI05871 (Doa)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0043880;
RRID:BDSC_43880
Genetic
reagent
(D. melanogaster)
MI08818 (qless)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0051110;
RRID:BDSC_51110
Genetic
reagent
(D. melanogaster)
MI06179 (DCX-EMAP)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0043047;
RRID:BDSC_43047
Genetic
reagent
(D. melanogaster)
MI14396 (CG6293)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0059511;
RRID:BDSC_59511
Genetic
reagent
(D. melanogaster)
MI00445 (Nlg3)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0031005;
RRID:BDSC_31005
Genetic
reagent
(D. melanogaster)
MI09222 (CG1578)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0051263;
RRID:BDSC_51263
Genetic
reagent
(D. melanogaster)
MI00494 (wnd)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015b
flybaseID#
_FBst0031028;
RRID:BDSC_31028
Genetic
reagent
(D. melanogaster)
MI02915 (Ask1)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0036163;
RRID:BDSC_36163
Genetic
reagent
(D. melanogaster)
MI03136 (LPCAT)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0036425;
RRID:BDSC_36425
Gnetic
reagent
(D. melanogaster)
MI10071 (Trpl)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0053455;
RRID:BDSC_53455
Genetic
reagent
(D. melanogaster)
MI09900 (Sap47)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#
_FBst0053794;
RRID:BDSC_53794
Genetic
reagent
(D. melanogaster)
MI01646 (CG1815)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0035948;
RRID:BDSC_35948
Genetic
reagent
(D. melanogaster)
MI04010 (Tbh)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0056660;
RRID:BDSC_56660
Genetic
reagent
(D. melanogaster)
MI13728 (CG17841)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0059189;
RRID:BDSC_59189
Genetic
reagent
(D. melanogaster)
MI05741 (CG1632)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0042106;
RRID:BDSC_42106
Genetic
reagent
(D. melanogaster)
MI10889 (CG17167)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0056092;
RRID:BDSC_56092
Genetic
reagent
(D. melanogaster)
MI00986 (CG32698)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0035095;
RRID:BDSC_35095
Genetic
reagent
(D. melanogaster)
MI15214 (CG13375)Venken et al. (2011a);
Nagarkar-Jaiswal et al., 2015a
flybaseID#_
FBst0060995;
RRID:BDSC_60995
Genetic
reagent
(D. melanogaster)
Nmnatywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the geneNmnat
Genetic
reagent
(D. melanogaster)
Stub1ywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene Stub1
Genetic
reagent
(D. melanogaster)
Ubqnywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene Ubqn
Genetic
reagent
(D. melanogaster)
Itp-r83Aywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene Itp-r83
Genetic
reagent
(D. melanogaster)
CG18769ywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene CG18769
Genetic
reagent
(D. melanogaster)
CG13390ywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene CG13390
Genetic
reagent
(D. melanogaster)
Med27ywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene Med27
Genetic
reagent
(D. melanogaster)
CG11679ywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene CG11679
Genetic
reagent
(D. melanogaster)
amxywing2+This studyfly strain carrying the
ywing2+ dominant marker
replacing the gene amx
Genetic
reagent
(D. melanogaster)
Nmnat::GFP::NmnatWTThis studyfly strain carrying the
Nmnat gene with S(GSS)4...
EGFP coding sequence…
(GSS)4 integrated internally
into the protein between
3R:24945353 and 3R:24945353
Genetic
reagent
(D. melanogaster)
Nmnat::GFP::NmnatW129GThis studyfly strain carrying the Nmnat
gene with S(GSS)4...EGFP
coding sequence…(GSS)4
integrated internally into
the protein between 3R:
24945353 and 3R:24945353
and bearing a mutation
producing W192G
Genetic
reagent
(D. melanogaster)
Nmnat::GFP::NmnatΔ251…257This studyfly strain carrying the Nmnat
gene with S(GSS)4...EGFP
coding sequence…(GSS)4
integrated internally into
the protein between 3R:
24945353 and 3R:24945353
and bearing a deletion
removing amino acids 251…257
Genetic
reagent (D. melanogaster)
Nmnat::GFP::NmnatC344S, C345SThis studyfly strain carrying the
Nmnat gene with S(GSS)4...
EGFP coding sequence…
(GSS)4 integrated internally
into the protein between 3R:
24945353 and 3R:24945353
and bearing a mutation
producing C344S, C345S
Recombinant
DNA reagent
Double HeaderThis studyRecombination Mediated
Cassette Exchange donor
plasmid containing SA-T2A
-GAL4-polyA and SA-GFP-SD
in the opposite orientation
Recombinant
DNA reagent
pattB ywing2+This studyVector for φC31
integrated transgenesis
that expresses the
yellow gene product in
the wings
Recombinant
DNA reagent
pattB ybody+This studyVector for φC31
integrated transgenesis
that expresses the
yellow gene product
in the body
Recombinant
DNA reagent
p{ywing2+}This studyDonor vector compatible
with Golden Gate cloning
carrying the yellow
wing2 + dominant marker
flanked by nucleotides
‘GG’ and ‘CC’ upstream
and downstream, respectively
Recombinant
DNA reagent
p{ybody+}This studyDonor vector compatible
with Golden Gate cloning
carrying the yellow body
dominant marker flanked
by nucleotides ‘GG’ and ‘C
C’ upstream and
downstream, respectively
Recombinant
DNA reagent
p{EGFP Donor}This studyDonor vector compatible
with Golden Gate cloning
carrying the EGFP coding
sequence flanked by (GSS)
linker sequences
Recombinant
DNA reagent
p{mCherry Donor}This studyDonor vector compatible
with Golden Gate cloning
carrying the mCherry
coding sequence flanked
by (GSS) linker sequences
Recombinant
DNA reagent
p{T2a-GAL4 Donor}}This studyDonor vector compatible
with Golden Gate cloning
carrying the T2a viral
peptide sequence in frame
with the GAL4 transcription
factor coding sequence
Recombinant
DNA reagent
p{T2a-GAL4-PolyA Donor}}This studyDonor vector compatible
with Golden Gate cloning
carrying the T2a viral
peptide sequence in frame
with the GAL4 transcription
factor coding sequence
terminating in the
SV40 transcriptional
terminator
Recombinant
DNA reagent
pCFD3-dU6:3gRNAPort et al. (2014)Addgene_
plasmid_#49410
Recombinant
DNA reagent
Double HeaderThis studyRecombination Mediated
Cassette Exchange donor
plasmid containing
SA-T2A-GAL4-polyA and
SA-GFP-SD in the
opposite orientation
Antibodyanti-GFP antibody
conjugated with FITC
AbcamRRID: AB_305635used at 1:500
Antibodyanti-GFPInvitrogenCat#_ A11122used at 1:500

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. The whole genome sequencing files are available on Zenodo (https://zenodo.org/record/1341241).

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

Sequences of oligonucleotides, sgRNAs and key vectors used in this study are shown along with a protocol for designing donor templates using the yellow wing2+ swappable insertion cassette.

https://doi.org/10.7554/eLife.38709.017
Transparent reporting form
https://doi.org/10.7554/eLife.38709.018

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