Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Podsumowanie

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Streszczenie

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments.

High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Wprowadzenie

As vectors of pathogens such as dengue¹, Zika², and chikungunya³ viruses, as well as malarial parasites⁴, mosquitoes represent a significant public health threat to humans. For all these diseases, there is a substantial focus of transmission intervention on the control of mosquito vectors. Study of the genes important in, for example, permissiveness to pathogens, mosquito fitness, survivorship, reproduction, and resistance to insecticides is key for developing novel mosquito control strategies. For such purposes, genome editing in mosquitoes is becoming a common practice, especially with the development of technologies such as HEs, ZFNs, TALENs, and most recently, CRISPR with Cas9. The establishment of gene-edited strains typically involves backcrossing individuals carrying the desired mutations for a few generations to minimize off-target and founder (bottleneck) effects, followed by crossing heterozygous individuals to generate homozygous or trans-heterozygous lines. In the absence of a dominant marker, molecular genotyping is necessary in this process because, in many cases, no clear phenotypic traits can be detected for heterozygous mutants.

Although sequencing is the gold standard for genotypic characterization, performing this across hundreds, or possibly thousands of individuals, poses significant costs, labor, and time required to obtain results, which is especially critical for organisms with short lifespans such as mosquitoes. Commonly used alternatives are Surveyor nuclease assay⁵ (SNA), T7E1 assay⁶, and high resolution melt analysis (HRMA, reviewed in⁷). Both SNA and T7E1 use endonucleases that cleave only mismatched bases. When a mutated region of the heterozygous mutant genome is amplified, DNA fragments from mutant and wild-type alleles are annealed to make mismatched double-stranded DNA (dsDNA). SNA detects the presence of mismatches via digestion with a mismatch-specific endonuclease and simple agarose gel electrophoresis. Alternatively, HRMA uses the thermodynamic properties of dsDNA detected by dsDNA-binding fluorescent dyes, with the disassociation temperature of the dye varying based on the presence and type of mutation. HRMA has been used for the detection of single-nucleotide polymorphisms (SNPs)⁸, mutant genotyping of zebra fish⁹, microbiological applications¹⁰, and plant genetic research¹¹, among others.

This paper describes HRMA, a simple method of molecular genotyping for mutant mosquitoes generated by CRISPR/Cas9 technology. The advantages of HRMA over alternative techniques include 1) flexibility, as it has been proven useful for various genes, a wide range of indel sizes, as well as the distinction between different indel sizes and heterozygous, homozygous, and trans-heterozygous differentiation^12,13,14, 2) cost, as it is based on commonly used PCR reagents, and 3) time-saving, as it can be performed in just a few hours. In addition, the protocol uses a small body part (a leg) as a source of DNA, allowing the mosquito to survive the genotyping process, permitting the establishment and maintenance of mutant lines.

Protokół

1. Scanning for single nucleotide polymorphisms (SNPs), HRMA primer design, and primer validation

1. SNP identification in wild-type laboratory colony mosquitoes
   1. Select the target exon to disrupt proper polypeptide translation.
      NOTE: The target should be close to the start codon or amongst key residues required for protein function. The shorter the exon (e.g., ≤200 bases), the more difficult it is to target and analyze. Avoid editing close to the boundaries of an exon, as this forces one of the HRMA primers to either cross an intron or be in an intron. This is undesirable because SNP rates tend to be much greater in those regions.
   2. Primer design
      1. Go to the NCBI Blast - Primer Blast website¹⁵, copy and paste the chosen exon in the box at the top of the page | choose the PCR product size (ensure it encompasses most of the exon) | choose the organism.
      2. Click on Advanced parameters | Opt (for PCR Product Tm) and add 60 (for an optimal temperature of 60 °C) | Get Primers. Keep the other parameters as default.
   3. Obtain genomic DNA (gDNA) from the wild-type laboratory colony. Set aside 10 mosquitoes, anesthetize them with CO₂, and place them in a Petri dish on ice to keep them inactive. Set up 10 tubes containing 0.5 µL of the reagent for release of DNA from tissue and 20 µL of dilution buffer, both provided in the DNA release kit suggested in the Table of Materials.
   4. Remove one leg from a single mosquito using forceps and place it in a corresponding tube of a diluted solution of the DNA-release reagent (from step 1.1.3), completely submerging the leg in the solution. Repeat this step with the remaining mosquitoes, wiping the forceps with 70% ethanol before proceeding to the next one.
   5. Incubate the leg-containing solution at room temperature for 2-5 min, then at 98 °C for 2 min, and allow it to cool down while setting up the PCR reaction.
      NOTE: Plates containing the released gDNA can be stored at -20 °C, and the protocol can be paused at this point.
   6. Prepare 10 tubes containing 10 µL of 2x PCR Master Mix, primers to a 0.5 µM final concentration, and molecular grade water to a final volume of 20 µL, and transfer 1 µL of the diluted sample to each tube. Perform PCR following these cycling parameters: 98 °C 5 min, 40 cycles of 98 °C for 5 s, 60 °C 30 s, 72 °C 20 s per kb; final extension of 72 °C for 1 min.
   7. Purify the PCR products with an enzyme to degrade the residual PCR primers and dephosphorylate excess dNTPs or any column clean-up kit. Proceed with sequencing the samples.
   8. Analyze each electropherogram for the presence of double peaks/ambiguous bases, and adjust base calls manually in each sequence using the appropriate degenerate base code.
      NOTE: This step must be performed before multiple sequence alignment as it is common for the base-calling software to select the more prominent peak as the "true" base call, giving the false impression of an absence of SNPs.
   9. Perform a multiple sequence alignment using the alignment software, SeqMan Pro, listed in the Table of Materials or other open-source alignment software, such as ClustalW¹⁶ or T-Coffee¹⁷.
      1. Open the alignment software | click on Add Sequences | select the desired sequences and click on Add | once all sequences are chosen, click on Done.
      2. Click on Assemble to perform the alignment. To open the alignment, click on Contig 1 and analyze the alignment and identify the SNPs (Figure 1).
         NOTE: As an alternative to steps 1.1.3-1.1.6, PCR can be performed from isolated genomic DNA obtained from bulk samples (derived from >10 individuals). Sequenced amplicons can be analyzed directly for the presence of SNPs appearing as double peaks in the electropherogram, though rare SNPs will be more difficult to detect.
   10. Design 3-5 single guide RNAs (sgRNAs), avoiding regions containing any SNP identified above, following the protocol described in¹⁸.
2. HRMA primer design
   1. Test the exon sequence for the possible formation of secondary structures during PCR using mFold¹⁹.
      1. Go to the UNAFold Web Server | click on mFold | on the dropdown menu, click on Applications | DNA Folding Form.
      2. Enter the sequence name in the box and paste the exon.
      3. Change the folding temperature to 60 °C; on the Ionic conditions, change [Mg⁺⁺] to 1.5 and on Units switch to mM; click on Fold DNA.
      4. On Output, below Structure 1, click on pdf to open the Circular Structure Plots. 
   2. Go to NCBI Blast - Primer Blast¹⁵ for primer design.
   3. Copy and paste the selected exon sequence determined by sequencing in step 1.1 (the sequence that contains the lowest number of SNPs or no SNPs) in the box at the top of the page.
   4. Use the symbol < > to mark and exclude sequences that contain SNPs, the target site, and regions with possible formation of secondary structure.
   5. Select the PCR product size to be between 80 and 150 bp and choose the organism.
      NOTE: Larger fragment sizes can be successfully used (~300 bp). However, longer amplicons may decrease sensitivity between sequences differing in one or just a few base pairs.
   6. Click on Get Primers. Select 2–3 pairs of primers to be tested (ideally, primer sites are ≥20–50 bp away from any CRISPR target sites).
3. Primer validation
   1. Perform a gradient PCR using gDNA from a single individual.
      1. Prepare a master mix and remove one sample for the non-template control (NTC) in a separate tube. Add the template to the remaining master mix and aliquot into a 96-well plate.
      2. Follow the cycling parameters: 98 °C 30 s, 34 cycles of 98 °C for 10 s, 55–65 °C 30 s, 72 °C 15 s; final extension of 72 °C for 10 min.
      3. Generate thermal melt profiles following the parameters: denaturation step 95 °C for 1 min, annealing 60 °C for 1 min, melt curve detection between 75 °C and 95 °C in 0.2 °C increments, with a hold time of 10 s at each temperature.
         NOTE: Only annealing temperatures with a single thermal melt profile should be used.
4. Proceed to generate the mutant lines with embryo injections as described in¹².

2. Preparation of genomic DNA from mosquito legs

1. Separate the G₁ mosquitoes by sex at the pupal stage so that they do not mate before genotyping, and make sure that age-matched, wild-type control mosquitoes will be available to be used for references and backcrosses. Sex-separate them likewise.
2. Gather the materials needed (Figure 2A) and prepare a 96 well PCR plate with 0.5 µL of the DNA-release reagent and 20 µL of dilution buffer (from the gDNA release kit) per individual to be genotyped (Figure 2B) and leave it on ice. Reserve two reactions for NTC.
3. Label a tray for mosquito vials (Drosophila vials) so each well on the 96-plate corresponds to the respective mosquito vial in the tray.
4. Anesthetize the G₁ mosquitos with CO₂ and place them in a glass Petri dish to keep them sedated (Figure 2C,D). Anesthetize and place 8 wild-type mosquitoes in a second Petri dish.
5. Wipe a pair of tweezers with 70% ethanol and remove one of the mosquito hind legs (Figure 2E,F).
6. Submerge the leg in the DNA-release reagent solution, place the mosquito in the corresponding vial, and close it with a sponge (Figure 2G,H).
7. Wipe the tweezers again with 70% ethanol and proceed with removing the leg from the next mosquito. Repeat steps 2.5-2.7 until the 96-well plate is completed.
   NOTE: It is important to wipe the tweezers with 70% ethanol. This minimizes DNA cross-contamination.
8. Seal the plate with an optical PCR plate seal (Figure 2I) and incubate the 96-well plate containing the legs at room temperature (RT) for 2-5 min and then at 98 °C for 2 min. Allow the plate to cool down to RT while preparing the PCR mix.
   NOTE: The entire process typically takes 3-4 h. If the mosquitoes must be kept in the vials for more time than the expected duration (especially ≥1 day), place a small piece of raisin (or source of sugar and water) with each mosquito to ensure the survival of the mosquitoes during extended incubations.

3. HRMA

1. Perform PCR.
   1. Prepare a master mix containing the following components per each reaction: 10 µL of 2x buffer (from the gDNA release kit), 0.5 µM of each primer, 1 µL of EvaGreen dye, 0.4 µL of polymerase (from gDNA release kit), and complete to 19 µL with molecular-grade water.
   2. Using a multichannel pipet, transfer 19 µL of the master mix into each well. Transfer 1 µL of the DNA release solution containing mosquito DNA prepared in section 2 to the plate (Figure 3A). Seal the plate with an optical PCR plate seal.
   3. Perform the PCR following the cycling parameters: 98 °C for 5 min, 39 cycles of 98 °C for 10 s, the chosen annealing temperature (72 °C) for 30 s; final extension 72 °C for 2 min.
2. Generate thermal melt profiles following the parameters: denaturation step 95 °C for 1 min, annealing 60 °C for 1 min, melt curve detection between 75 °C and 95 °C in 0.2 °C increments, with a hold time of 10 s at each temperature. See the Supplemental Material and Supplemental Figure S1-S5 for a detailed description of the software setup for HRMA run using the CFX96 Real-Time System.
3. Examine the melt profiles (Figure 3B). Assign wild-type control to the reference cluster.
   NOTE: The software (see the Table of Materials) automatically normalizes the data and designates clusters with colors for different melt curves.
4. Mark the different clusters with corresponding colors on the 96-well template (Figure 3C).
5. Select the individuals with curves of interest, remove them from the tubes, and backcross (Figure 3D). Blood-feed the mated females and collect G₂ eggs.

4. Sequence verification by Sanger sequencing

1. Purify the PCR product from the wells with selected mosquitoes (from the plate prepared in section 3.1), using an enzyme to degrade the residual PCR primers, and dephosphorylate excess dNTPs. Alternatively, use any column clean-up kit and proceed with sequencing the samples.
   NOTE: Direct sequencing may be more challenging when the PCR product size is small (≤200 bp). In such cases, design primers to amplify larger fragments encompassing the target site (≥250 bp) and amplify by PCR using the DNA in the 96-well plate (step 2.2).
2. Analyze and identify the indels using trace viewer software (Figure 4). Alternatively, Poly Peak Parser software²⁰ can help detect the mutation in heterozygous individuals.
   1. Go to the Poly Peak Parser website, select the sequence from the mutant individuals on the Browse menu, and copy and paste the reference sequence on the box.
      NOTE: The alignment between the alternate allele and the reference will appear automatically on the right side of the screen.

Wyniki

Mosquitoes containing mutations in the genes AaeZIP11 (putative iron transporter²¹) and myo-fem (a female-biased myosin gene related to flight muscles¹³) were obtained using CRISPR/Cas9 technology, genotyped using HRMA, and sequence-verified (Figure 5). Figure 5A and Figure 5C show the normalized fluorescence intensity from the HRM curves from AaeZIP11 and

Dyskusje

High-resolution melt analysis offers a simple and fast solution for the identification of indels generated by CRISPR/Cas9 technology in the vector mosquito Ae. aegypti. It provides flexibility, enabling the genotyping of mosquitoes mutated for a wide range of genes from flight muscle to iron metabolism and more^13,14. HRMA can be performed in just a few hours from sample collection to the final analyses. Additional time is required for primer design and w...

Materiały

Name	Company	Catalog Number	Comments
70% Ethanol			70% ethanol solution in water
96-well PCR and Real-time PCR plates	VWR	82006-636	For obtaining genomic DNA (from the mosquito leg)
96-well plate templates			House-made printed, for genotype recording
Bio Rad CFX96	Bio Rad		PCR machine with gradient and HRMA capabilities
Diversified Biotech reagent reservoirs	VWR	490006-896
Exo-CIP Rapid PCR Cleanup Kit	New England Biolabs	E1050S
Glass Petri Dish	VWR	89001-246	150 mm x 20 mm
Hard-shell thin-wall 96-well skirted PCR plates	Bio-rad	HSP9665	For HRMA
Multi-channel pipettor (P10)	Integra Biosciences	4721
Multi-channel pipettor (P300)	Integra Biosciences	4723
Nunc Polyolefin Acrylate Sealing tape, Thermo Scientific	VWR	37000-548	To use with the 96-well  PCR plates for obtaining genomic DNA
Optical sealing tape	Bio-rad	2239444	To use with the 96-well skirted PCR plates for HRMA
Phire Animal tissue direct PCR Kit (without sampling tools)	Thermo Fisher	F140WH	For obtaining genomic DNA and performing PCR
Plastic Fly Vial Dividers	Genesee	59-128W
Precision Melt Analysis Software	Bio Rad	1845025	Used for genotyping the mosquito DNA samples and analyzing the thermal denaturation properties of double-stranded DNA (see protocol step 3.3)
SeqMan Pro	DNAstar Lasergene software		For multiple sequence alignment
Single-channel pipettor	Gilson
Tweezers Dumont #5 11 cm	WPI	14098
White foam plugs	VWR	60882-189
Wide Drosophila Vials, Polystyrene	Genesee	32-117
Wide Fly Vial Tray, Blue	Genesee	59-164B