CRISPR-Cas9 Mediated Genome Editing in Bicyclus anynana Butterflies

CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation.


Introduction
In the past three decades, discovery and engineering of tools such as zinc finger nucleases (ZFNs), transcription-activator like effective nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized the field of genome engineering [1,2].
Zinc finger nucleases were the first-generation tools for genome engineering. The ability of zinc finger domains to recognize specific DNA sequences was first described in Xenopus laevis [3]. In 1996, Kim et al. reported the development of a fusion protein where they managed to link zinc finger domains to the cleavage domain of a FokI endonuclease. The resulting dimers showed the ability to cleave DNA at specific sites [4]. Zinc finger nucleases have been utilized to edit genomes of a wide array of model organisms including fruit flies [5,6], silk moths [7], zebrafish [8,9], Arabidopsis [10], rats [11] and pigs [12] and non-model organisms such as the cricket Gryllus bimaculatus [13] and the butterfly Danaus plexippus [14]. Although ZFNs showed great promise, the difficulty in designing and testing ZFNs and the associated costs restricted the use of this technology to only a few labs [1,15,16].
The next wave of genome engineering involved the hybrid proteins TALENS. Scientists studying phytopathogenic bacteria Xanthomonas in 2009 discovered transcription activator-like effectors (TALEs) proteins, capable of recognizing specific sites of DNA [17]. The following year, another group of researchers developed a new class of hybrid proteins called TALENs, which contains the site-specific TALEs fused to the catalytic site of the FokI endonuclease [18]. TALENs are much easier to synthesize, have higher sequence specificity compared to ZFNs, and do not require tedious validations [15,19]. This second-generation tool led to rapid acceleration in genome engineering and was used to study model organisms such as fruit flies [20], silk moths [21,22] nematodes [23], zebrafish [24], pigs [25] and frogs [26], as well as non-model organisms such as mosquitos Anopheles gambiae [19] and Aedes

Experimental Stages
The major experimental steps involved in CRISPR-Cas9 mediated gene manipulation in Bicyclus anynana include: (1) Synthesis and verification of sgRNA; (2) Injection of the sgRNA-Cas9 mixture into embryos; (3) Rearing of hatched individuals, and (4) Genotyping of mutant individuals (Table 1). Figure 1 illustrates the workflow and below we provide a brief explanation of what is entailed in each of these steps. Detailed protocols then follow.   Each sgRNA contains 100 nucleotides where 20 bases contain the recognition site that mediates the hybridization with target DNA, and the remaining 80 bases are involved in sgRNA-Cas9 hybridization [59]. The 20 bases are going to be target specific, whereas the remaining 80 bases are conserved across all experiments. The sgRNA is synthesized using ultramers (long DNA sequences) with the forward primer consisting of 64 bases and reverse primer 80 bases. A double-stranded sequence of the sgRNA (sgDNA) is synthesized using high fidelity polymerase and is later used as a template for synthesizing sgRNA using in vitro transcription. After purification of sgRNA, the efficacy of sgRNA-Cas9 hybrid is verified via an in vitro cleavage assay. The presence of multiple bands in the treated sample ensures that the sgRNA is functional.

Injection of the sgRNA-Cas9 Mixture into Embryos
In this step, sgRNA and Cas9 protein (or mRNA) are mixed together along with Cas9 buffer and non-toxic food dye (for visualizing the amount of sgRNA-Cas9 injected). The RNA-protein hybrid can also be stored at 4 • C or −20 • C for future use. The mixture is injected using fine glass needles at optimal pressure. This step requires good injection skills and proper tools and materials to prepare fine needles (see procedure for detailed information).

Rearing of Hatched Individuals
Rearing hatched larvae and adults requires proper humidity (80%), temperature (27 • C), and lighting (12-12 day-night cycle). Larvae are reared on young corn leaves and adults on mashed bananas.

Genotyping of Mutant Individuals
This step involves screening of mutants. Individuals should be monitored for mosaic clones at every stage. After screening, genomic DNA is extracted from mutant tissue and the sequence of interest is amplified using polymerase chain reaction (PCR). A T7 cleavage assay is carried out on the amplified DNA to verify whether sequence variants are present in the targeted sequence. Once sequence variants are confirmed, the amplified DNA is cloned in bacteria and sequenced. The sequences are aligned, and sites of indels are identified.     Figure 3) with orientation 5′ to 3′ (+) without the PAM sequence (Note: sequences closer to the 5′ region of the gene of interest and having high GC content should be preferred) and insert it in the grey highlighted part of the template below:  Identify the DNA sequence to be modified. Two versions of the B. anynana genome are available on lepbase [60]. Genes can be searched using the search field.

2.
Copy the DNA sequence and go to the webpage mentioned in [59]. Paste the sequence in the 'Paste a nucleotide sequence' box.

4.
Copy a candidate target sequence (see Figure 3) with orientation 5 to 3 (+) without the PAM sequence (Note: sequences closer to the 5 region of the gene of interest and having high GC content should be preferred) and insert it in the grey highlighted part of the template below: This is the forward primer for guide synthesis.
The reverse primer for sgRNA synthesis is the same for all target sites (see below):

-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTG
CTATTTCTAGCTCTAAAAC-3 .   Table 4 in a 200 μL PCR tube:  Add reagents as mentioned in Table 4 in a 200 µL PCR tube: 3.1.2. Synthesis of sgDNA 1. Add molecular grade water to the lyophilized primers in their original tubes (forward and reverse) to make a stock solution of 100 mM. Prepare a working solution of 10 mM by combining 10 μL of the stock solution with 90 μL molecular grade water in a new set of 1.5 mL tubes. 2. Add reagents as mentioned in Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Use high fidelity DNA polymerase only. Prepare at least three tubes for each sgDNA to obtain enough yield.

3.
Setup the PCR reaction with the conditions mentioned in Table 5:  1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop. PAUSE STEP: Prepare a working concentration of 500 ng/μL. The purified DNA can be stored at 4 °C for over one month. For long-term storage, use a −20 °C freezer.
3.1.4. In Vitro Transcription to Prepare sgRNA 1. Add the reagents mentioned in Table 6 in a 1.5 mL microcentrifuge tube: 1.
Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s.

2.
Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through.

3.
Add 500 µL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time.

4.
Spin the moist column for one additional min at 13,000 rpm and discard the collection tube.

5.
Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 µL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6.
Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop.  1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop.
PAUSE STEP: Prepare a working concentration of 500 ng/μL. The purified DNA can be PAUSE STEP: Prepare a working concentration of 500 ng/µL. The purified DNA can be stored at 4 • C for over one month. For long-term storage, use a −20 • C freezer.
Add the reagents mentioned in Table 6 in a 1.5 mL microcentrifuge tube: Incubate the mixture in water bath at 37 • C for 15 min. 5.
Remove 1 µL of the reaction mixture in a 200 µL PCR tube. Add 7 µL of molecular grade water and 2 µL of 2× RNA loading dye. 6.
Heat the sample at 70 • C for 10 min and run it in 1% agarose gel.  Table 4 in a 200 μL PCR tube:  Table 5:

Setup the PCR reaction with the conditions mentioned in
CRITICAL STEP: RNA degrades very fast. To prevent degradation properly, clean the gel tray using MilliQ water, use fresh buffer and run the gel for 15-20 min at low voltage.
Add 80 µL of molecular grade water to the reaction tube from the previous step to raise the volume to 100 µL.

3.
Vortex the mixture for 10 s and store at −20 • C for 15-20 min.

5.
Carefully remove the supernatant.  Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Be very careful not to disturb the pellet. 6.
Dry the sample in a vacuum concentrator and add 20 µL of molecular grade water. 7.
Prepare a stock concentration of 600 ng/µL by adding additional water (after a Nanodrop reading) and store aliquots at −20 • C.  1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop. PAUSE STEP: Prepare a working concentration of 500 ng/μL. The purified DNA can be stored at 4 °C for over one month. For long-term storage, use a −20 °C freezer.
3.1.4. In Vitro Transcription to Prepare sgRNA 1. Add the reagents mentioned in Table 6 in a 1.5 mL microcentrifuge tube: 1. Add 10 µg of Addgene plasmid #46757 (pT3TS-nCas9n), 2 µL of restriction enzyme and molecular grade water (to make up the volume to 20 µL) in a 1.5 mL microcentrifuge tube.
Add the reagents mentioned in Table 7 in a 1.5 mL centrifuge tube: For poly(A) tailing add the reagents mentioned in Table 8 to the tube above: Table 8. Reaction mixture of Cas9-mRNA poly(A) tailing. Centrifuge at 4 • C, 14,000 rpm for 15 min. 3.

Reagents
Carefully remove the supernatant and resuspend the pellet in 100 µL 70% ethanol. 4.
Remove the supernatant and dry the sample in a vacuum concentrator. 6.
Resuspend the pellet in 10 µL molecular grade water and measure the concentration using Nanodrop. Store the RNA at −20 • C.  1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop.   Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Carefully remove the gut material from the larvae as it might contaminate the DNA sample. One larva should be enough for a yield of around 500 ng/µL in 100 µL volume. Alternatively, tissues can be extracted from the thorax of adult Bicyclus or embryos.

2.
Add 200 µL TL buffer and homogenize the tissue in homogenizer using 0.5 mm stainless steel beads for 5 min.

3.
Add 20 µL OB protease solution and vortex for 10 s.

4.
Incubate the mixture in shaking heat block at 55 • C for 16 h.

5.
Centrifuge the tube at 14,000 rpm for 5 min to precipitate the cell debris. 6.
Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube and add 220 µL BL buffer. 7.
Incubate the mixture in water bath at 70 • C. 8.
Transfer the mixture to HiBind DNA column and centrifuge at 14,000 rpm for 1 min. 10. Discard the filtrate and add 500 µL HBC buffer. 11. Centrifuge at 14,000 rpm for 30 s and discard the filtrate. 12. Transfer the column to a fresh 2.0 mL collection tube. 13. Add 500 µL DNA wash buffer and centrifuge at 14,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 14. Centrifuge the empty column at 14,000 rpm for 1 min and transfer the column into a fresh 1.5 mL microcentrifuge tube. 15. Add 100 µL elution buffer or molecular grade water to the column and let it sit for 5 min at room temperature. 16  1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop. PAUSE STEP: Prepare a working concentration of 500 ng/μL. The purified DNA can be stored at 4 °C for over one month. For long-term storage, use a −20 °C freezer.
3.1.4. In Vitro Transcription to Prepare sgRNA 1. Add the reagents mentioned in Table 6 in a 1.5 mL microcentrifuge tube: Resuspend the lyophilized primers using molecular grade water to make a stock solution of 100 ng/µL. Prepare a working solution of 10 mM as described above.

2.
Add the reagents mentioned in Table 9 in a 200 µL PCR tube:   Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Prepare at least five tubes in order to identity the most optimal annealing temperature in a gradient PCR reaction.

3.
Setup the gradient PCR reaction with conditions as mentioned in Table 10:    Table 6 in a 1.5 mL microcentrifuge tube: Transfer the reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s.

2.
Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through.

3.
Add 500 µL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time.

4.
Spin the moist column for and additional min at 13,000 rpm and discard the collection tube.

5.
Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 µL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6.
Centrifuge at 13,000 rpm for 1 min and measure the concentration using Nanodrop.    Table 6 in a 1.5 mL microcentrifuge tube:  Add the reagents mentioned in Table 11 in a 1.5 mL microcentrifuge tube: 1.
Add the reagents mentioned in Table 12 in a 1.5 mL microcentrifuge tube: Turn on the needle puller machine and set a program with the settings as mentioned in Table 13: 2.
Arrest the glass capillary in place and click on the enter button. Wait for the process of heating and pulling the glass to be completed.

3.
Carefully remove the needles from the machine and place them on plasticine (as shown in Figure 4). 3. Carefully remove the needles from the machine and place them on plasticine (as shown in Figure 4).   Remove the leaves and collect the embryos in a paper cup.

3.
Prepare a Petri dish with thin strips of double-sided tape attached to the bottom of the plate. 4.
Using a paintbrush, carefully arrange the embryos on the double-sided tape ( Figure 5). 3. Carefully remove the needles from the machine and place them on plasticine (as shown in Figure 4).   Table 14 in a 1.5 mL microcentrifuge tube: Add the reagents mentioned in Table 14 in a 1.5 mL microcentrifuge tube: Pipette 3 µL of the sgRNA-Cas9 mix using a 20 µL Microloader tip and transfer the content to the needle by filling it from the back.

3.
Attach the needle to the injection holder and break the tip of the needle (e.g., remove the molten glass at the tip that obstructs the opening) by gently pressing against the side of a Petri dish under a dissecting microscope.  Table 4 in a 200 μL PCR tube:  Table 5: CRITICAL STEP: Be careful while breaking the needle tip. If the desired sharpness is not attained, transfer the mixture back to the 1.5 mL tube (by pushing it out with air pressure) and repeat the steps above.

4.
Inject the embryos under a dissecting microscope until the mixture is visible inside the embryos.

Rearing Hatchling and Screening for Mutants. Time for Completion: 4-5 Weeks
Rearing of the Hatchlings (Condition for Rearing Include 12-12 Day-to-Night Cycle, Temperature of 27 • C, and Humidity of 80%).

1.
Place a wet cotton ball inside the Petri plate above and incubate at 27 • C. It will take 3-4 days for the embryos to hatch (use the unhatched embryos for the T7 endonuclease assay).

2.
Transfer the hatched larvae into a paper cup with one young corn leaf. Keep feeding these larvae inside cups with fresh cut leaves until they reach the 3rd instar. Transfer these older larvae to larger rearing cages. Keep note of any changes in phenotype and image these abnormal individuals/tissues.

3.
Pupae are assigned to separate paper or plastic cups, individually, where adults emerge after one week.

4.
Freeze the adults at −20 • C for imaging and genotyping.  Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Carefully remove the gut material from the larvae to minimize contaminating the larval DNA with bacterial DNA.

2.
Add 200 µL TL buffer and homogenize the tissue in homogenizer using 0.5 mm stainless steel beads for 5 min.

3.
Add 20 µL OB protease solution and vortex for 10 s.

4.
Incubate the mixture in a shaking heat block at 55 • C for 16 h.

5.
Centrifuge the tube at 14,000 rpm for 5 min to precipitate the cell debris. 6.
Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube and add 220 µL BL buffer. 7.
Incubate the mixture in a water bath at 70 • C. 8.
Transfer the mixture to a HiBind DNA column and centrifuge at 14,000 rpm for 1 min. 10. Discard the filtrate and add 500 µL of HBC buffer. 11. Centrifuge at 14,000 rpm for 30 s and discard the filtrate. 12. Transfer the column to a fresh 2.0 mL collection tube. 13. Add 500 µL DNA wash buffer and centrifuge at 14,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 14. Centrifuge the empty column at 14,000 rpm for 1 min and transfer the column into a fresh 1.5 mL microcentrifuge tube. 15. Add 100 µL elution buffer or molecular grade water to the column and let it sit for 5 min at room temperature. 16. Centrifuge at 14,000 rpm for 1 min and measure the concentration using Nanodrop. Store the DNA at 4 • C for immediate use.    Table 6 in a 1.5 mL microcentrifuge tube: Add the reagents mentioned in Table 15 in a 200 µL PCR tube:   Table 4 in a 200 μL PCR tube:  Table 5: CRITICAL STEP: Prepare at least five tubes to identify the most optimal annealing temperature using gradient PCR.

2.
Setup the PCR reaction with the conditions mentioned in Table 16:    Table 6 in a 1.5 mL microcentrifuge tube: Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s.

2.
Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through.

3.
Add 500 µL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time.

4.
Centrifuge the empty column for one additional min at 13,000 rpm and discard the collection tube.

5.
Transfer the column to a fresh 1.5 mL microcentrifuge tube and add 20 µL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6.
Centrifuge at 13,000 rpm for 1 min and measure the concentration using Nanodrop.
Methods Protoc. 2018, 1, x FOR PEER REVIEW 10 of 31 1. Transfer the completed reaction volume to a 1.5 mL microcentrifuge tube and add an equal volume of binding buffer. Vortex the mixture for 5 s. 2. Transfer the mixture to the GeneJET PCR purification column and centrifuge at 13,000 rpm for 30 s. Discard the flow through. 3. Add 500 μL of wash buffer and centrifuge at 13,000 rpm for 30 s. Discard the flow through and repeat this step one more time. 4. Spin the moist column for one additional min at 13,000 rpm and discard the collection tube. 5. Transfer the column to a new 1.5 mL microcentrifuge tube and add 20 μL of elution buffer or molecular grade water. Incubate the column at room temperature for 3-5 min. 6. Centrifuge at 13,000 rpm for 1 min and measure the concentration of the elution using Nanodrop.
PAUSE STEP: Prepare a working concentration of 500 ng/μL. The purified DNA can be stored at 4 °C for over one month. For long-term storage, use a −20 °C freezer.
3.1.4. In Vitro Transcription to Prepare sgRNA 1. Add the reagents mentioned in Table 6 in a 1.5 mL microcentrifuge tube:  Prepare two 200 µL PCR tubes and add the reagents mentioned in Table 17 into each of them: 2. Perform T7 hybridization in thermocycler with the conditions mentioned in Table 18:

3.
Add 1 µL T7 endonuclease in one tube and incubate both tubes in water at 37 • C for 15 min.

4.
Run the samples in 1% agarose gel for 30 min.
3.6.5. Cloning of amplified DNA fragments (using pGEM-T Vector System)

1.
Add the reagents mentioned in Table 19 in a 1.5 mL microcentrifuge tube (ligation mixture): Take out one vial of competent cells and keep the tube on ice for 15 min.

4.
Transfer 5 µL of ligation mixture into the competent cell tube and tap gently to mix the solution.

5.
Leave the mixture on ice for 30 min. 6.
Heat shock the cells by transferring the tube into a water bath at 42 • C for 45 s.  Table 4 in a 200 μL PCR tube:  Table 5: CRITICAL STEP: Be careful not to exceed the heat shock step above 45 s. 7.

Setup the PCR reaction with the conditions mentioned in
Transfer the tube into ice and leave it for 2 min. 8.
Add 500 µL of autoclaved LB broth and incubate the cells in bacterial incubation chamber at 37 • C with shaking speed of 225 rpm for 2 h. 9.
Centrifuge the tube at 3000 rpm for 4 min. 10. Inside a biological safety cabinet add the reagents mentioned in Table 20 to an LB agar plate: Table 20. Reagents for the screening of positive bacterial colonies.

Reagents Volume (µL)
IPTG 25 X-GAL 25 Ampicillin 25 11. Spread the reagents on the plate using glass beads and let the plate dry inside the hood. 12. Add 50 µL of supernatant from step 9 and spread across the plate using the glass beads. 13. Once dried, seal the plate using parafilm and incubate the plate inside a bacterial incubator at 37 • C for 14 h.
3.6.6. Colony PCR on Transformed Clones 1. In a 1.5 mL microcentrifuge tube, add 10 µL molecular grade water. Pick a transformed white colony and transfer it into the tube. Vortex gently to homogenize the colony.   Table 4 in a 200 μL PCR tube: 3. Setup the PCR reaction with the conditions mentioned in Table 5: CRITICAL STEP: Prepare at least 10 clones (colonies) for testing.
Setup the PCR reaction with the conditions mentioned in Table 22:

4.
Run the reaction mixture in a 1% agarose gel for 30 min and note down the colonies with a single band of the expected size, e.g., those that don't have an empty plasmid.

5.
Inside a laminar hood, add 5 µL of ampicillin stock solution into a test tube with 5 mL LB broth. Transfer 5 µLs of homogenized cells from step 1. Do this step for every positive colony. 6.
Incubate the tubes in a bacterial incubation chamber at 37 • C and 225 rpm for 14-16 h.

1.
Harvest the cells in a 1.5 mL centrifuge tube at 3000 rpm for 5 min (pellet can be stored in 40% glycerol at −80 • C for future use).

2.
Discard the supernatant and resuspend the pellet in 250 µL of resuspension buffer.

3.
Add 250 µL of lysis buffer and mix by inverting the tube 6-10 times.
Centrifuge at 14,000 rpm for 5 min and transfer the supernatant to GeneJET spin column. 6.
Centrifuge the column at 14,000 rpm for 30 s. 7.
Add 500 µL of wash buffer and centrifuge at 14,000 rpm for 30 s. Discard the flow through and repeat this step one more time.
Transfer the column to a 1.5 mL microcentrifuge tube and add 20 µL of elution buffer or molecular grade water. Incubate the mixture at room temperature for 3 min.   Add the reagents mentioned in Table 23 in a 200 µL PCR tube: Add the reagents mentioned in Table 24 in another 200 µL PCR tube:

2.
Setup sequencing PCR reaction with the conditions mentioned in Table 25: 3. In 1.5 mL microcentrifuge tubes, add 40 µL molecular grade water and transfer the reaction mix from the previous step.
Carefully remove the supernatant and dry the sample in a vacuum concentrator. 9.
Store at −20 • C until sequencing.
3.6.9. Analyzing the Sequencing Results and Determination of Indel Sites

1.
Copy the sequences in a word file with specific identifiers.

2.
Align the sequences along with the original DNA sequence using ClustalW [63] or the Geneious multiple sequence alignment tool.

3.
Look for the indels at the expected site and save the file for future use.

Expected Results
The results are explained with reference to the gene Engrailed 1 (En1). The sequence of the complete transcript is given below ( Figure 6) where bases in red represent the 5 and 3 UTRs, respectively: 3. Look for the indels at the expected site and save the file for future use.

Expected Results
The results are explained with reference to the gene Engrailed 1 (En1). The sequence of the complete transcript is given below (Figure 6) where bases in red represent the 5′ and 3′ UTRs, respectively:

Gel Electrophoresis of sgDNA
A band at 150 bp should be visible with 100-200 ng of the sgDNA product (see Figure 7).
3. Look for the indels at the expected site and save the file for future use.

Expected Results
The results are explained with reference to the gene Engrailed 1 (En1). The sequence of the complete transcript is given below (Figure 6) where bases in red represent the 5′ and 3′ UTRs, respectively:

Gel Electrophoresis of sgRNA
A band at 100 bp should be visible with 500 ng of the sgRNA product (see Figure 8). A band at 100 bp should be visible with 500 ng of the sgRNA product (see Figure 8). TROUBLESHOOTING: Make sure that all the tubes and pipette tips are RNase free. Use an RNase blaster solution to remove RNase contamination.

Preparation and Purification of Cas9 mRNA
Yield of Cas9 mRNA. The expected yield of Cas9 mRNA after purification should be around 15-40 μg. The concentration of Cas9 mRNA obtained in this experiment was 15 μg. The expected yield of genomic DNA from the epidermal tissue of one 5th instar larvae is around 50 μg.

Gel Electrophoresis of Amplified DNA
Band at 783 bp should be visible with 100-200 ng of the PCR product with En1_E1_5′_F and En1_E1_5′_R primers (see Figure 9).  The expected yield of genomic DNA from the epidermal tissue of one 5th instar larvae is around 50 µg.

Gel Electrophoresis of Amplified DNA
Band at 783 bp should be visible with 100-200 ng of the PCR product with En1_E1_5 _F and En1_E1_5 _R primers (see Figure 9). A band at 100 bp should be visible with 500 ng of the sgRNA product (see Figure 8). TROUBLESHOOTING: Make sure that all the tubes and pipette tips are RNase free. Use an RNase blaster solution to remove RNase contamination.

Preparation and Purification of Cas9 mRNA
Yield of Cas9 mRNA. The expected yield of Cas9 mRNA after purification should be around 15-40 μg. The concentration of Cas9 mRNA obtained in this experiment was 15 μg. The expected yield of genomic DNA from the epidermal tissue of one 5th instar larvae is around 50 μg.

Gel Electrophoresis of Amplified DNA
Band at 783 bp should be visible with 100-200 ng of the PCR product with En1_E1_5′_F and En1_E1_5′_R primers (see Figure 9).

Yield of Amplicon
The yield of amplified DNA from 5 tubes of PCR reaction mixture was 5.6 µg. The yield usually varies in between 4 and 10 µg.

Gel Electrophoresis of sgRNA-Cas9 Cleaved DNA Fragment
The cleavage assay should yield two smaller bands (see Figure 10 lane 4). The size of smaller bands denotes the site of cleavage and can be cross-verified by looking at the original template sequence and the cas9 cleavage site originally designed (see Figure 2).

Yield of Amplicon
The yield of amplified DNA from 5 tubes of PCR reaction mixture was 5.6 μg. The yield usually varies in between 4 and 10 μg.

Gel Electrophoresis of sgRNA-Cas9 Cleaved DNA Fragment
The cleavage assay should yield two smaller bands (see Figure 10 lane 4). The size of smaller bands denotes the site of cleavage and can be cross-verified by looking at the original template sequence and the cas9 cleavage site originally designed (see Figure 2).

Thickness of Needle
The expected thickness of needle after pulling is shown in Figure 11. After breaking the tip, the diameter should be approximately 0.01-0.05 mm.

Thickness of Needle
The expected thickness of needle after pulling is shown in Figure 11. After breaking the tip, the diameter should be approximately 0.01-0.05 mm.

Hatching Rate
The hatching rate usually depends on the gene targeted. For En1, the hatch rate was between 5% and 50%. Table 26 shows the number of hatched individuals using 100 ng/μL and 300 ng/μL of En1_G2 sgRNA.

Hatching Rate
The hatching rate usually depends on the gene targeted. For En1, the hatch rate was between 5% and 50%. Table 26 shows the number of hatched individuals using 100 ng/µL and 300 ng/µL of En1_G2 sgRNA.

Number of Mutants
In experiments 1-4, we were unable to obtain any mutants. In experiment 5, we observed six individuals during the larval stage with segmentation defects and three adult individuals with eyespot and venation defects ( Figure 12).

Number of Mutants
In experiments 1-4, we were unable to obtain any mutants. In experiment 5, we observed six individuals during the larval stage with segmentation defects and three adult individuals with eyespot and venation defects ( Figure 12).

Yield of DNA from Mutant Clones
The yield of DNA from 1 larva was 2.4 μg. Expected yield is around 2-4 μg (total yield) depending on the amount of tissue used for isolation.

Yield of DNA Amplicon
The expected yield of amplified DNA is around 4 μg.

Gel Electrophoresis after T7 Endonuclease Assay
The T7 endonuclease assay ( Figure 13) should yield smaller bands equivalent to the cleavage assay shown in Figure 10.  The yield of DNA from 1 larva was 2.4 µg. Expected yield is around 2-4 µg (total yield) depending on the amount of tissue used for isolation.

Yield of DNA Amplicon
The expected yield of amplified DNA is around 4 µg.

Gel Electrophoresis after T7 Endonuclease Assay
The T7 endonuclease assay ( Figure 13) should yield smaller bands equivalent to the cleavage assay shown in Figure 10.

Number of Mutants
In experiments 1-4, we were unable to obtain any mutants. In experiment 5, we observed six individuals during the larval stage with segmentation defects and three adult individuals with eyespot and venation defects ( Figure 12).

Yield of DNA from Mutant Clones
The yield of DNA from 1 larva was 2.4 μg. Expected yield is around 2-4 μg (total yield) depending on the amount of tissue used for isolation.

Yield of DNA Amplicon
The expected yield of amplified DNA is around 4 μg.

Gel Electrophoresis after T7 Endonuclease Assay
The T7 endonuclease assay ( Figure 13) should yield smaller bands equivalent to the cleavage assay shown in Figure 10.

Number of Colonies
We obtained around 200 blue and white colonies after plating 50 µL of bacterial supernatant cultured for 2 h. Expected number of colonies is between 100 and 300.

Yield of Plasmid
From 2 mL of bacterial culture grown overnight, we obtained 2.4 µg plasmid. The yield varies depending on the amount of culture used (usually between 2 and 4 µg).

Sequencing Alignment Result
The sites of indels can be identified by aligning the sequences and analyzing the expected target site of CRISPR-Cas9 ( Figure 14).

Sequencing Alignment Result
The sites of indels can be identified by aligning the sequences and analyzing the expected target site of CRISPR-Cas9 ( Figure 14). Figure 14. Insertion of nucleotides in the target site (sequence 1 is from the wild type). The sgRNA recognition sequence is marked by the red box. 1. In a 2 L beaker, add 500 mL MilliQ water and the reagents specified in Table 27. Table 27. Reagents for 50× TAE buffer preparation.

Reagents
Weight/Volume Trizma base 242 g Disodium EDTA 18.61 g Glacial Acetic Acid 57.1 mL

1.
In a 20 mL measuring cylinder add 0.2 g of X-Gal and raise the volume to 10 mL using DMSO.

2.
Mix the content and make 1 mL aliquots in 1.5 mL microcentrifuge tubes. Store the content at −20 • C.

1.
Add the reagents mentioned in Tables 28 and 29 in 200 mL conical flasks: For LB broth (100 mL)  LB broth tubes can be stored at room temperature or 4 • C for up to two months.

4.
Prepare LB agar plates by transferring 20 mL LB agar into Petri plates inside biological safety cabinet. 5.
LB agar plates can be stored at 4 • C for up to two months.
Author Contributions: T.D.B. and A.M. conceived the experiments and wrote the manuscript; T.D.B. designed and performed the experiment, analyzed the data, and developed the associated videoarticle.