Genetics and Genomics

Expanding the Drosophila toolkit for dual control of gene expression

Jonathan Zirin author has email address
Barbara Jusiak
Raphael Lopes
Ben Ewen-Campen
Justin A. Bosch
Alexandria Risbeck
Corey Forman
Christians Villalta
Yanhui Hu
Norbert Perrimon author has email address

Dept of Genetics, Harvard Medical School, Boston, MA
Dept of Physiology and Biophysics, University of California, Irvine, CA
Howard Hughes Medical Institute, Boston, MA

https://doi.org/10.7554/eLife.94073.1

Open access
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Figures and data

Strategy for CRISPR knock-in of T2A-LexA-GAD and T2A-QF2.
(A) Donor vectors for knock-in. pHDR-T2A-LexA-GAD-Hsp70-3xP3-RFP and pHDR-T2A-QF2-Hsp70-3xP3-RFP contain T2A-LexA-GAD or QF2 transcriptional activators followed by HSP70 terminators. pHDR-T2A-QF2-T2A-LexA-GAD-3xP3-RFP contains both activators flanked by FRT sites followed by SV40 terminator. All the vectors contain a 3XP3-RFP transformation marker flanked by loxP sites. (B) Long homology arm cloning method. ∼1000bp homology arms are amplified from genomic DNA and inserted into the AscI and SacI sites by Gibson Assembly. A separate target-gene-specific guideRNA is cloned into U6 promoter expression vector such as pCFD3. (C) Drop-in cloning method. Based on the gRNA-int200 method previously described (Kanca et al., 2022). A company synthesizes and clones a DNA fragment into the pUC57_Kan_gw_OK2 vector. The resulting plasmid contains the following elements: 1) two guide RNAs under the control of a U6 promoter, one (gRNA1) targeting the vector (pink arrows) to linearize the homology donor in vivo, and another (gRNA^geneX) targeting the gene of interest; 2) a tRNA sequence to allow liberation of the individual guides by the endogenous tRNA processing machinery; 3) 200 bp short homology arms; and 4) BbsI and SacI cloning sites. The AscI/SacI T2A-LexA-GAD, T2A-QF, or T2A-LexA-GAD-T2A-QF fragments can then be ligated in a single directional cloning step into the BbsI/SacI sites to produce the donor plasmid. (D) T2A-LexA-GAD and (E) T2A-QF2 knock-in strategy. CRISPR based HDR causes integration of the T2A-LexA-GAD or T2A-QF2 in the most 5’ coding exon common to all or most isoforms, resulting in expression of the activators under control of the endogenous gene regulatory region. The knock-in also produces a truncated endogenous protein and thus a strong loss-of-function allele.

T2A-LexA-GAD and T2A-QF2 knock-in lines

Tissue specificity of T2A-LexA-GAD and T2A-QF2 knock-ins.
(A-KK) T2A-LexA-GAD knock-in lines crossed to a LexAop-GFP reporter and T2A-QF2 knock-in lines crossed to a QUAS-GFP reporter. Panels show 3^rd instar larva. GFP shows the driver line expression pattern. RFP shows the 3XP3 transformation marker, which labels the posterior gut and anal pads of the larva. Gene names and tissues are on the left. Note that most of the lines are highly tissue-specific and are comparable between the LexA-GAD and QF2 knock-ins. (LL-MM) 3^rd instar imaginal disc from the insertions in the nubbin (nub) gene. Insertions in the daughterless gene (da) and nub are the exception, as the T2A-LexA-GAD, but not the T2A-QF2, gives the expected expression pattern.

3XP3-RFP can cause misexpression of T2A-LexA-GAD or T2A-QF2.
(A) T2A-QF2-3XP3-RFP in the repo gene crossed to a QUAS-GFP reporter. In 3^rd instar larva, the reporter is expressed in the expected glial cells, but also mis-expressed in gut and anal pad (yellow asterisk). (B) T2A-QF2 in the repo gene with the 3XP3-RFP removed by Cre-Lox recombination, crossed to a QUAS-GFP reporter. Removal of 3XP3-RFP eliminated gut and anal pad misexpression and did not affect glial cell expression.

T2A-QF2-T2A-LexA-GAD double driver lines.
(A) CRISPR based HDR strategy for integration of the T2A-QF2-T2A-LexA-GAD-3XP3 in the most 5’ coding exon common to all or most isoforms, resulting in expression of both activators under control of the endogenous gene regulatory region. The knock-in also produces a truncated endogenous protein and thus a strong loss-of-function allele. If desired, one of the two coding regions can then be excised with Flp, resulting in flies that express only QF2 or LexA-GAD. (B) Alternative strategy allows gene enhancers to be cloned upstream of of T2A-QF2-T2A-LexA-GAD. The vector backbone includes an attB site for phiC31 insertion into attP flies. (C-D) T2A-QF2-T2A-LexA-GAD knock-ins crossed to a QUAS-GFP + LexAop-mCherry double reporter line. (C) The elav^{T2A-QF2-T2A-LexA-GAD}knock-in drives both QUAS-GFP and LexAop-mCherry in the larval brain. There is some leakiness of mCherry in the body wall muscle (arrowheads). (D) The hh^{T2A-QF2-T2A-LexA-GAD} knock-in drives both QUAS-GFP and LexAop-mCherry in the posterior of the wing imaginal disc. GFP expression is much less than mCherry in the wing pouch (asterisks). (E-F) enhancer-T2A-QF2-T2A-LexA-GAD lines crossed to a QUAS-GFP + LexAop-mCherry double reporter line. (E) The dpp-blk enhancer-T2A-QF2-T2A-LexA-GAD line drives both QUAS-GFP and LexAop-mCherry along the anterior/posterior boundary of the wing imaginal disc. GFP expression is much less than mCherry in the wing pouch (stars). (F) The Ilp2 enhancer-T2A-QF2-T2A-LexA-GAD line drives both QUAS-GFP and LexAop-mCherry in the insulin-producing cells of the larval brain (arrows). The fat body mCherry expression is also present in the reporter stock and does not indicate LexA-GAD activity.

Generation of single drivers from T2A-QF2-T2A-LexA-GAD knock-ins by hs-FLP.
(A) FLP/FRT recombination scheme. Flies containing both hs-FLP and a T2A-QF2-T2A-LexA-GAD knock-in are heat shocked during larval development to induce one of two mutually exclusive recombination events in their germline between either FRT or FRT3. (B) heat shock of hsFLP; hh^{T2A-QF2-T2A-LexA-GAD}and hsFLP; dpp^{T2A-QF2-T2A-LexA-GAD} flies produces frequent recombinants, both T2A-QF2 and T2A-LexA-GAD. The bar graph shows the proportion of heat-shocked animals that produced at least one recombinant offspring. The dot plot shows the proportion of recombinant offspring per heat-shocked parent. Mean +/-SD is indicated. (C-D) Validation of individual hh^T2A-QF2 and hh^T2A-LexA-GAD derivatives by immunofluorescence. All panels show 3^rd instar larval wing discs dissected from potential hh^{T2A-QF2-T2A-LexA-GAD}recombinants crossed to a QUAS-GFP + LexAop-mCherry reporter line. (C) Wing disc from non-recombinant hh^{T2A-QF2-T2A-LexA-GAD} showing expression of both GFP and mCherry in the posterior of the wing disc. (D) Wing disc from recombinant hh^T2A-QF2 showing expression of GFP but not mCherry in the posterior of the wing disc. (E) Wing disc from recombinant hh^T2A-LexA-GAD showing expression of mCherry but not GFP in the posterior of the wing disc. Validation of (F) hh^T2A-QF2 and hh^T2A-LexA- ^GAD derivatives and (G) dpp^T2A-QF2 and dpp^T2A-LexA-GADderivatives by PCR from genomic DNA from individual flies. In all panels, for brevity, T2A-QF2-T2A-LexAop, T2A-QF2 and T2A-LexAop, are notated as Q+L, Q(-L) and L(-Q), respectively.

TRiP LexAop and QUAS shRNA vectors produce effective gene knockdown.
(A) shRNAs for knockdown or genes for overexpression were cloned into pLexAop-WALIUM20 and pQUAS-WALIUM20, derived from the TRiP WALIUM20 vector. (B-J) Dorsal view of adult fly thoraces resulting from crosses of LexAop or QUAS shRNAs to da^T2A-LexA-GAD (generated in this study), Tub-LexA-GAD (BDSC 66686), or Tub-QF2 (BDSC 51958). (B-C) white shRNA control produced no thoracic phenotypes in any of the crosses. (E-G) forked shRNA produced a forked bristles phenotype. (H-J) ebony shRNA produced a darkened cuticle phenotype. The da^T2A-LexA- ^GAD driver produced the strongest phenotype (compare panel H to I and J).

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