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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Abstract

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

Introduction

The genetic toolbox for targeted gene expression has been well developed in Drosophila, making it one of the best model systems to investigate the function of genes involved in a wide variety of cellular processes. Binary expression systems, such as yeast Gal4/UAS (upstream activation sequence), was first adopted for tissue-specific enhancer trapping and gene misexpression in the Drosophila genetic model1 (Figure 1). This system facilitated the development of a large number of techniques such as spatiotemporal regulation of gene overexpression, misexpression, knockout in selected groups of cells as well as in cell ablation, cell marking, live tracing of cellular and molecular processes in embryo and tissues, lineage tracing and mosaic analyses during development. A number of binary transcription system, such as the bacterial LexA/LexAop system (Figure 1) and Neurospora Q-system, are powerful genetic tools that are now widely used in Drosophila, in addition to the original Gal4/UAS system for targeted gene expression1,2,3.

Here, we present a method to generate highly reliable tissue-specific binary expression system by employing a genome-editing technique. The recent advancements in CRISPR/Cas9 genome editing technology have allowed unprecedented opportunities to make directed genome changes in a broad range of organisms. Compared to the other available genome editing techniques, the CRISPR/Cas9 system is inexpensive, efficient, and reliable. This technology utilizes components of the bacterial adaptive immune system: a Cas9 endonuclease of Streptococcus pyogenes that creates a double-strand break (DSB), and a chimeric guide RNA (gRNA), which guides the Cas9 to a particular genome site for targeted DSB4. The cells contain the machinery to repair the DSB using different pathways. Non-homologous end joining (NHEJ) leads to small insertions or deletions to disrupt gene function, while homology-directed repair (HDR) introduces a defined directed/desirable genomic knock-in/knock-out by using an exogenous HDR donor as a template. The HDR-based replacement strategy can efficiently be utilized to generate a highly reliable tissue-specific binary expression system, which can overcome all the limitations of the traditional enhancer trap methods. We describe a step-by-step procedure for utilization of CRISPR/Cas9-based HDR repair in generating a binary transcription driver line that is expressed under the control of endogenous transcriptional and post-transcriptional regulation of a Drosophila gene. In this protocol, we demonstrate the generation of a driver line specific for branchless (bnl) gene encoding an FGF family protein that regulates branching morphogenesis of tracheal airway epithelium5. In this example, the first coding exon of the bnl gene was replaced by the sequence of a bacterial LexA transactivator sequence without altering any endogenous cis-regulatory sequences of the bnl gene. We show that the strategy generated a bnl-LexA driver line that spatiotemporally controls the expression of a reporter gene placed downstream of LexAoperator (LexAop or LexO) exclusively in bnl-expressing epithelial/mesenchymal/neuronal cells.

Protocol

1. Designing and Constructing the gRNA Expression Vector

  1. To precisely replace a long defined region of an exon, use a dual gRNA approach6, in which each gRNA can specifically target two ends of the selected region of interest. To obtain an accurate gene-specific spatiotemporal expression of the driver, select two gRNA target sites within the first coding exon of the gene.
  2. For Drosophila melanogaster, select the gRNA target sites using the flyCRISPR Optimal Target Finder tool (http://tools.flycrispr.molbio.wisc.edu/targetFinder/). Other available CRISPR design tools can be used as well, including the Optimized CRISPR Design tool (http://crispr.mit.edu), FlyCas9 (https://shigen.nig.ac.jp/fly/nigfly/cas9), and E-CRISP (www.e-crisp.org/E-CRISP).
    NOTE: The following protocols of gRNA design and cloning was adopted from methods previously described6,7.
    1. Copy the actual sequence into the box provided in the online tool. Select the most recently released Drosophila melanogaster genome annotation version, “r_6,” in the drop-down menu for “Select genome.” In the field “Select guide length (nt),” input“20.” Select to find “All CRISPR targets” and click “Find CRISPR Targets”.
    2. Evaluate all the candidate gRNA targets by setting “Maximum” for “Stringency” and “NGG Only” for “PAM”. Try to select the gRNA sites without any potential off-targets or a minimum number of off-targets possible. Use the web-tool described in Doench et al. 2014 8 to select gRNAs with a potential “good” activity score.
    3. Simultaneously, ensure that there is no single nucleotide polymorphism in the gRNA targets in the genome of the parent fly line selected for injection (e.g., nos-Cas9 on X chromosome, BL# 54591). Follow the protocol described in Gratz et al. 20157 to extract the genomic DNA from the parent fly line. PCR amplify, approximately, the 500–1,000 nt regions using primers that flank the sequence of interest and a high-fidelity DNA polymerase; sequence-verify the PCR amplified products.
  3. For generating a tandem gRNA expression vector, follow a ligation-independent cloning protocol6 to introduce two protospacer sequences into a pCFD4 RNA expression vector. Note that an improved multi-gRNA expression vector (pCFD5) is now available where both the sgRNAs are expressed from the strong U6:3 promoters9 (https://www.crisprflydesign.org). Use a DNA Assembly method to clone the gRNAs into the pCFD4 vector.
    1. Design and order the forward and reverse primers for introducing two protospacer sequences into the RNA expression vector pCFD4 (Table 1A). As previously described6, the forward primer contains the first protospacer sequence flanked by regions corresponding to the U6-1 promoter and gRNA core in pCFD4; the reverse primer contains the reverse complement sequence of the second protospacer flanked by regions corresponding to the gRNA core and U6-3 promoter in pCDF4.
    2. Resuspend primers to 100 μM with DNase and RNase-free double distilled water (ddH2O). Make a 10 μM working concentration primer. Use pCFD4 plasmid as a template and set up PCR reaction using a high-fidelity polymerase and recommended settings for PCR reaction6.
    3. To clone the amplified product into pCFD4, digest pCFD4 plasmid with BbsI enzyme by setting up the following reaction: 2–5 μg of pCFD4 plasmid, 5 μL of 10x digestion buffer, 1 μL of BbsI enzyme, and ddH2O to bring the final volume to 50 μL. Mix the reaction contents by gently tapping the tube and collecting all the scattered droplets from the wall of the tube to the bottom by a brief spin. Incubate the reaction mix at 37 °C for 2 h.
    4. Run the PCR product from step 2 and the digested product from step 3 on a 1% agarose gel. Perform the electrophoresis for enough time to completely separate the DNA bands. Cut the correct sized DNA bands under a UV trans-illuminator before purifying the expected products using gel elution column following the manufacturer’s instruction (see Table of Materials). The PCR product should be 600 bp, and the linearized pCFD4 plasmid should be ~6.4 kb 6.
    5. Set up the DNA assembly reaction in a PCR tube, per the manufacturer’s instruction (see Table of Materials).
    6. Transform 2 μL of the assembly product into competent bacteria (DH5α or similar strains with ΔrecA1, ΔendA1), plate on Ampicillin (100 μg/mL) containing LB (LB/Amp) agar plates, and incubate at 37 °C overnight. Note that the DNA assembly mix might be toxic to certain bacteria strains, but diluting the assembly mix can reduce the toxicity.
    7. Pick 3–4 ampicillin-resistant colonies from the overnight-incubated plate, inoculate the colony in 3 mL LB containing 100 μg/mL ampicillin, and grow inoculated bacteria in a 37 °C shaker overnight. Extract plasmid from the overnight-cultured bacteria using the plasmid mini-prep kit following the manufacturer’s instruction (see Table of Materials).
    8. Sequence the plasmids with T3 universal primer to screen for the correct clones containing the tandem gRNA insertion. Establish a glycerol stock of bacteria transformed with a sequence-verified pCFD4-gRNA in 20% glycerol and store in a -80 °C freezer for future use.

2. Designing and Constructing the HDR Donor

  1. For genomic knock-in of a Gal4 or LexA sequence, design a double-stranded HDR donor containing the transactivator sequence flanked by two homology arms.
    1. Use >1.5 kb left and right homology arms flanking the gRNA targeting site(s). Extended homology arms increase the efficiency of HDR during the repair process 10.
    2. PCR amplify the homology arms from the genomic DNA (gDNA) extracted from the parent fly line selected for injection (e.g., nos-Cas9 (on X chromosome), BL# 54591). Use a proof-reading hot-start Taq-DNA polymerase enzyme suitable for long PCR extension (see Table of Materials). Sequence-verify.
  2. To avoid retargeting of the gRNA to the engineered locus, design the replacement donor in such a way that the gRNA recognition sites are disrupted by the exogenous sequence introduced.
    NOTE: to avoid altering the putative cis-regulatory elements, always select the gRNA recognition sites within the coding exon region.
  3. For generating a replacement cassette, plan the DNA Assembly strategy to join four segments together: the 5’ and 3’ homology arms, the middle exogenous transactivator DNA sequence, and the linearized cloning vector backbone (e.g., pUC19 or other commonly used cloning vectors). Following the manufacturer’s guideline for DNA assembly (see Table of Materials), design the suitable primers for PCR amplification and assembly of each segment. Resuspend primers to 100 μM with ddH2O. Make a 10 μM working concentration primer. Use high-fidelity polymerase for all the PCR reactions. Follow the following protocol:
    1. PCR amplify a Gal4 or LexA expression cassette from an available vector (e.g., nls-LexA:p65; the ideal Gal4/LexA/QF2 expression plasmids can be found and obtained from common resources such as Addgene) using the primers listed in Table 1B.
      1. To retain all the original transcriptional and post-transcriptional regulations of the replaced exon on the expression of the transactivator DNA sequence, design the cassette in such a way that the edited genomic allele would express a chimeric mRNA of the transactivator and the gene. Preserve the 5’ and 3’ end of the targeted exon to retain any splicing signal.
      2. Incorporate a T2A self-cleaving peptide sequence between the residual 5’ coding exon and the transactivator sequence to prevent translation into a chimeric protein. Add a translation stop codon after the exogenous transactivator sequence (Figure 5).
    2. PCR amplify the homology arms from the genomic DNA (gDNA) extracted from the parent fly line selected for injection (e.g., nos-Cas9 (on X chromosome), BL# 54591) using the primers listed in Table 1B. Use high-fidelity polymerase for all the PCR reactions using the systems as follows: 5 μL of 5x Reaction Buffer, 0.5 μL of 10 mM dNTPs, 1.25 μL of 10 μM Forward Primer, 1.25 μL of 10 μM Reverse Primer, 0.5 μL of Template DNA, 0.25 μL of High-Fidelity DNA Polymerase, 16.25 μL of ddH2O.
    3. Use linearized cloning vector such as pUC19. A number of donor vectors are now available. See a comprehensive list at the following website- http://flycrispr.molbio.wisc.edu.
    4. Run the PCR products on a 1% agarose gel. Perform the electrophoresis for enough time to ensure a clear separation of the desired band from the undesired non-specific bands (if any). Gel-purify the expected DNA fragments. Measure the concentration of each purified DNA fragment using a spectrophotometer.
    5. Set up the DNA assembly reaction following the manufacturer’s instruction. Mix the linearized cloning vector and target fragments from step 3 and step 4 in the following reaction: 1 μL of linear cloning vector (50 ng/μL), 2–3 fold excess of each target fragment, 10 μL of 2x DNA assembly master mix, bring the final reaction volume to 20 μL with ddH2O. Incubate the reaction mix for 1 hour at 50 °C.
    6. Transform 2 μL of the assembly product into competent bacteria, plate on LB/Amp agar plates containing 100 μg/mL ampicillin. Also, add X-Gal + IPTG on the plate for blue/white screening.
    7. Pick 3–4 white colonies from the overnight-incubated plate, inoculate the colony in 3 mL LB containing 100 μg/mL ampicillin, and grow at 37 °C overnight in a shaker. Extract plasmid from the overnight-cultured bacteria using the plasmid mini-prep kit following the manufacturer’s manual (see Table of Materials).
    8. Screen the positive colony by PCR or restriction digestion.
    9. Sequence verify the HDR donor region of the final plasmid. Save a bacterial stock transformed with the correct clones in 20% glycerol at -80 °C for future inoculation.

3. Embryo Injection, Fly Genetics and Screening for Genome Editing

  1. Prepare the high purity endotoxin-free gRNA expression vector as well as the HDR donor plasmid using a plasmid maxiprep kit (see Table of Materials).
  2. Co-inject the gRNA expression plasmid (100 ng/μL) and the replacement donor (500 ng/μL) into the germline of nos-Cas9 embryos6. Note: we use a commercial service for injection, but this procedure can be performed in the laboratory as well.
  3. Fly genetics and screening (Figure 2 and Figure 3):
    1. When the injected embryos develop into adults, cross each single G0 flies to balancer flies. Select suitable balancer for the chromosome containing the targeted allele.
    2. Anesthetize the F1 offspring from each G0 cross on a CO2 pad and randomly pick 10-20 males under a stereomicroscope. Cross them individually to the balancer females as shown in Figure 3.
    3. When the F2 larvae hatch, pick the single F1 father from each cross and extract gDNA using the single fly genomic DNA preparation protocol:
      1. Prepare gDNA extraction buffer: 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, store at room temperature. Prepare 20 mg/mL Proteinase K stock solution and store in the freezer.
      2. Put each fly in a 1.5 mL micro-centrifuge tube and label the tube. Keep in the -80 °C freezer overnight.
      3. Prepare a fresh working volume of gDNA extraction buffer containing 200 µg/mL final concentration of Proteinase K.
        Note: Do not use an old buffer for this step.
      4. Squish each fly for 10–15 s with a pipette tip containing 50 μL of squishing buffer without dispensing the liquid. Dispense the remaining buffer into the tube and mix well. Incubate at 37 °C for 20–30 min.
      5. Put tubes in 95 °C heat block for 1–2 min to inactivate the Proteinase K.
      6. Spin down for 5 min at 10,000 x g. Store the preparation at 4 °C for further PCR analysis.
  4. Use the same method to prepare gDNA from a nos-Cas9 fly, which serves as a negative control. Perform three-step PCR based screens to identify the correct “ends out” HDR (Figure 2, Figure 5) using gDNA of each F1 male as a template5. Use 1 μL of the DNA prep in the following PCR reaction system: 10 μL of 2x PCR Master Mix with Dye, 1 μL of each primer (10 μM), 1 μL of DNA template, and 7 μL of ddH2O.
  5. As shown in Figure 5A, perform PCR using primers fwd1 and rev1 to screen for the existence of the insertion or replacement; perform PCR using fwd2 and rev2 primers to verify the insertion or replacement from 3’ region; perform PCR using primers M13F and rev3 to check “ends-in” HDR (Table 1C).
  6. Keep the fly lines with the confirmed ends-out HDR and establish balanced stocks from the F2 generation. Outcross to the balancer flies again to remove any unintended mutations on other chromosomes.
  7. Prepare high-quality gDNA from the established stocks for amplifying long PCR (>800–1,000 nt) amplicon with high-fidelity Taq polymerase and fully sequence verify the amplicons obtained from the engineered genomic regions. Alternatively, use crude gDNA extract as described earlier to amplify shorter (<800 nt), overlapping PCR products to sequence-validate the edited genome. Use the following protocol to prepare the good quality Drosophila genomic DNA:
    1. Put about 25 adult flies in a 1.5 mL microcentrifuge tube, and freeze in -20 °C or -80 °C freezer for at least 1 hour.
    2. Add 250 μL of Solution A (pH 9.0 Tris HCl 0.1 M, EDTA 0.1 M, SDS 1%).
    3. Homogenize the flies using the homogenizing pestles in microcentrifuge tubes, and put the tube on ice.
    4. Incubate at 70 °C for 30 min.
    5. Add 35 μL of KOAc (5 M), shake, and mix well, but do not vortex.
    6. Incubate on ice for 30 min.
    7. Spin at 12,000 x g for 15 min.
    8. With a 1 mL micropipette, carefully transfer only the clear supernatant to a new tube, leaving back any precipitate or interphase.
    9. Add 150 μL of isopropanol to the supernatant. Gently mix by inversion.
    10. Spin at 10,000 x g for 5 min.
    11. Carefully remove the supernatant and leave the pellet in the tube.
    12. Wash the pellet with 1 mL 70% EtOH.
    13. Spin at 12,000 x g for 5 min.
    14. Remove the supernatant. Air dry the pellet for 15 to 20 min. Do not over dry the pellet.
    15. Dissolve the pellet in 100 μL of ddH2O.
    16. Use high fidelity DNA polymerase suitable for long amplicon (>800 bp) to amplify the complete edited region. Ideally, take two primers outside the inserted cassette to amplify the genomic region. Gel purify the PCR product, and as described earlier, sequence verify the product with multiple overlapping primers.
  8. Validate the correct spatiotemporal expression patterns from dissected tissues/embryos of the engineered fly lines:
    1. Perform RT-PCR from the total RNA to verify the expression of the hybrid mRNA product (Table 1D).
    2. Perform an in situ hybridization of the mRNA product of the interest in the larval tissues/embryo to validate the expression.
    3. To screen for the accurate tissue-specific spatiotemporal expression patterns among the sequence-verified ends-out lines, cross each of the edited lines obtained for the binary expression driver with the LexO-GFP or LexO-RFP (for LexA driver) line (available from the Bloomington stock centers) and observe the LexA or Gal4 driven reporter-gene expression in embryo, larval, and adult tissues under a fluorescence microscope.

Results

This protocol was successfully used to generate a targeted binary expression reporter system specific for bnl expressing cells5. The cis-regulatory elements (CREs) that control complex spatiotemporal bnl expression are not characterized. Therefore, to achieve spatiotemporal expression under the control of the endogenous bnl regulatory sequence, only the first coding exon of bnl was designed to be replaced with the sequen...

Discussion

Traditionally, Drosophila enhancer traps were generated by two different methods. One of the ways includes random insertion of a driver (eg., Gal4) sequence in the genome by transposition (e.g., P-element transposition)1 . Alternatively, the driver sequences can be placed under the transcriptional control of a putative enhancer/promoter region in a plasmid construct, which would then be integrated into an ectopic site of the genome3,

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

We thank Dr. F. Port, Dr. K. O'Connor-Giles, and Dr. S. Feng for discussions on CRISPR strategy; Dr. T.B. Kornberg, and the Bloomington Stock Center for reagents; UMD imaging core facility; and funding from NIH: R00HL114867 and R35GM124878 to SR.

Materials

NameCompanyCatalog NumberComments
X-Gal/IPTGGentrox (Genesee Scientific)18-218cloning
LB-AgarBD DifcoBD 244520cloning
Tris-HClSigma AldrichT3253Molecular Biology
EDTASigma AldrichE1161Molecular Biology
NaClSigma AldrichS7653Molecular Biology
UltraPure DNase/RNase-Free WaterThermoFisher Scientific10977-023Molecular Biology
10%SDSSigma Aldrich71736Molecular Biology
KOAcFisher-ScientificP1190Molecular Biology
EtOHFisher-Scientific04-355-451Molecular Biology
GeneJET MiniprepThermoFisher ScientificK0503Miniprep
PureLink HiPure Plasmid Maxipep kitsThermoFisher ScientificK210006Maxiprep
BbsINEBR0539SRestriction enzyme
PrimersIDT-DNAPCR
pCFD4Kornberg LabDNA template and vector for gRNA
KAPA HiFi Hot Start- (Kapa Biosystems)Kapa biosystemsKK2601PCR
Q5-high fidelity TaqNEBNEB #M0491PCR
Gibson Assembly Master MixNEBNEB #E2611DNA assembly
pBPnlsLexA:p65UwAddgeneDNA template for LexA amplification
Proteinase KThermoFisher Scientific25530049Molecular Biology
2x PCR PreMix, with dye (red)SydlabMB067-EQ2RMolecular Biology
Gel elution kitZymo Research (Genesee Scientific)11-300Molecular Biology
TRI reagentSigma-AldrichMolecular Biology
Direct-zol RNA purification kitsZymo Research (Genesee Scientific)11-330Molecular Biology
OneTaq One-Step RT-PCR KitNEBE5315SMolecular Biology
lexO-CherryCAAXKornberg LabFly line
UAS-CD8:GFPKornberg labFly line
btl-Gal4Kornberg labFly line
MKRS/TB6BKornberg labFly line
Confocal Microscope SP5XLeicaImaging expression pattern
CO2 stationGenesee Scientific59-122WCUfly pushing
Stereo microscopeOlympusSZ-61fly pushing
Microtube homogenizing pestlesFisher-Scientific03-421-217genomic DNA isolation
NanoDrop spectrophotometerThermoFisher ScientificND-1000DNA quantification

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Tissue specificBinary Transcription SystemsDrosophilaGenome EditingCRISPR Cas9FGF HomologBranchlessTracheal Airway EpitheliumSpatiotemporal ExpressionTrans ActivatorGAL4LexAUASLexOEndogenous RegulationGuide RNAPCFD4 Vector

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