Methods xxx (2014) xxx–xxx

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CRISPR/Cas9 mediated genome engineering in Drosophila Andrew Bassett ⇑, Ji-Long Liu ⇑ Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, United Kingdom

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Drosophila melanogaster CRISPR Cas9 Genome engineering Targeted mutagenesis RNA injection

a b s t r a c t Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect novel mutations in desired target genes in Drosophila melanogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. The small insertion and deletion mutations that result from inefficient non-homologous end joining at this site are detected by high resolution melt analysis of whole flies and individual wings, allowing stable lines to be made within 1 month. Ó 2014 Published by Elsevier Inc.

1. Introduction Our ability to design DNA binding factors with exquisite specificity for desired target sequences has heralded a new wave of genome engineering techniques that allow targeted modifications of the genome to be achieved in many organisms [1–14]. This new genome engineering technology will enable more directed and elegant experiments to be performed to analyse structural and functional aspects of the genome. The CRISPR/Cas9 system was discovered as a bacterial defence system against invading viral pathogens, which uses fragments of RNA from the virus to target cleavage of the viral DNA through complementary base pairing [15–20]. This system has recently been shown to be active in other systems, including mammals [1–3], insects [6–12] and plants [13], and can be easily modified to target double strand breaks (DSB) at any desired target sequence by supplying it with a short guide RNA that is complementary to the target site within the DNA. The endogenous system involves three components. The Cas9 protein is an endonuclease that binds to a structure within a trans-acting CRISPR RNA (tracrRNA). The tracrRNA base pairs with a CRISPR RNA (crRNA), the first 20 nt of which determine the specificity of the Cas9 endonuclease. A simplified two component system has been described that fuses the tracrRNA and crRNA into a single synthetic guide RNA (sgRNA), making delivery of the components easier [1,2,18].

Abbreviations: CRISPR, clustered regularly interspaced palindromic repeats; Cas, CRISPR associated; DSB, double strand break; NHEJ, non-homologous end joining; HR, homologous recombination. ⇑ Fax: +44 1865 282849 (A. Bassett), +44 1865 285862 (J.-L. Liu). E-mail addresses: [email protected] (A. Bassett), [email protected] (J.-L. Liu).

The DSBs produced can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), and both can be useful to introduce mutations into the underlying DNA [21]. NHEJ repair is error prone, and often results in small insertions or deletions (indels) at the cut site, that can be mutagenic. Targeting two DSBs can also result in the deletion of intervening sequences, to generate longer deficiencies [6]. Induction of a DSB also enhances rates of HR repair, which can be used to enhance gene targeting efficiencies by several orders of magnitude [22–24]. This system has been developed for use in many organisms, including Drosophila, where multiple methods of introducing the Cas9 and sgRNA components have been developed [6–12] (Table 1). The Cas9 protein can be introduced by injection of mRNA or an expression vector into the early embryo [6–8], or by using a transgenic strain that produces the Cas9 protein under a germline-specific or ubiquitous promoter [9–11]. The sgRNA itself can be produced by in vitro transcription [7,8], or expressed from a pol III promoter derived from the U6 snRNA gene [6,9–11]. The use of a pol III promoter avoids capping and polyadenylation of the transcript, which may inhibit its activity. Again, in vitro transcribed sgRNA or an expression plasmid can be injected into Drosophila embryos, or transgenic strains can be produced that express the sgRNA ubiquitously. These techniques can be used in different combinations, and each has advantages in certain circumstances, or for specific experiments (Table 1). For instance, the highest reproducibility and efficiency of mutagenesis can be achieved by crossing two transgenic lines, but it relies on generating a transgenic line expressing each desired sgRNA, which is relatively time consuming. Although giving good mutagenesis efficiency, all of the techniques involving transgenic Cas9 expression rely on injection or crossing to the transgenic fly lines, making it difficult to compound mutations with pre-existing alleles, or inject into

http://dx.doi.org/10.1016/j.ymeth.2014.02.019 1046-2023/Ó 2014 Published by Elsevier Inc.

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A. Bassett, J.-L. Liu / Methods xxx (2014) xxx–xxx

Table 1 Comparison of CRISPR/Cas9 techniques in Drosophila. Reference

Gratz et al. [6]

Bassett et al. [7]

Yu et al. [8]

Kondo and Ueda [9]

Sebo et al. [11]

Ren et al. [10]

Cas9 promoter Cas9 delivery

hsp70 DNA injection U6

T7 mRNA injection T7

Sp6 mRNA injection

nos Transgenic

vasa Transgenic

nos Transgenic

T7

U6

U6

DNA injection Yellow

sgRNA injection Yellow, white

sgRNA injection

Transgenic

Yellow, K81, CG3708, CG9652, kl-3, light, RpL15

6–66 5.9–20.7

4–88 0–79

35.7–80 35.7–100

White, neuropeptide genes (Ast, capa, Ccap, Crz, Eh, Mip, npf), mir-219, mir-315 N/A 0–100

DNA injection EGFP, mRFP

U6a, U6b, nosmini DNA injection

0.25–1.37 1 month Yes

0–34.5 1 month Yes

2.1–98.9 1 month Yes

0–99.4 2–3 months No

sgRNA promoter sgRNA delivery Target genes

Mosaic G0 (%)a Germline mutants (among fertile flies) (%)b F1 mutant overall (%)c Overall Timescaled Applicable to all genetic backgroundse

White

N/A 35–71

N/A 0–100

7.7–24.7 1 month No

0–74.2 1 month No

This table is modified from Table 1 in Bassett and Liu [37]. a Percentage of flies that exhibit mosaic expression in the injected generation, either visibly in males or detected using HRMA (high resolution melt analysis). b Proportion of fertile flies giving rise to at least one mutant offspring. c Total number of mutant G1 offspring as a percentage of the total offspring. d Approximate overall timescale including the time spent generating transgenic fly stocks (if applicable). e All of the techniques involving transgenic delivery of Cas9 rely on injecting into specific fly lines, limiting their ability to compound mutations with existing lines, or generating mutations in other genetic backgrounds or Drosophila strains. N/A, not applicable to this technique, since Cas9 is germline restricted.

different genetic backgrounds. The described technique has the advantage that it can be performed in essentially any genetic background, and there is no possibility of integration of DNA constructs into the genome, but does require care in the production and handling of the injected RNA. Here we describe a detailed methodology to produce and inject mRNA encoding the Cas9 protein, and in vitro transcribed sgRNAs that can result in high efficiencies of mutagenesis of desired target genes by inefficient NHEJ. Up to 88% of flies have mosaic mutations in the target gene, which can be transmitted to up to 34.5% of total F1 offspring [7] (Table 1). We also describe the application of high resolution melt analysis (HRMA) to provide a simple and effective system of detection of the resulting indel mutations to enable generation of stable mutant lines [7]. This technique utilises the fact that indel mutations change the melting temperature of PCR products spanning the target site to rapidly and accurately detect mosaic and heterozygous mutant flies. 2. Materials and methods 2.1. Overview sgRNAs are designed to target the gene of interest that minimise potential off target effects and maximise mutagenic efficiency, and templates for their transcription are generated by a simple PCR. The sgRNA and mRNA encoding the Cas9 protein are generated by in vitro transcription, purified and coinjected into Drosophila embryos of essentially any genotype. Mosaic flies are identified by HRMA, and heterozygous mutant offspring from these flies are selected by analysis of PCR products from single wings by HRMA and sequencing. These flies are used to make stable stocks that can be used for further analysis. An overview of the process with approximate timings is shown in Fig. 1, and reagents required are listed in Supplementary Table 1. 2.2. sgRNA design 2.2.1. Target site choice Cas9 is guided to 20 nt target sequences in the genome that are complementary to the 50 end of the sgRNA, and these sequences

must be followed by an NGG protospacer adjacent motif (PAM) sequence (Fig. 2A). The PAM sequence does not appear in the sgRNA, but is nevertheless required by the Cas9 protein for efficient endonucleolytic cleavage of the DNA. These sequences can be on either strand of the DNA, making the expected frequency of such sites approximately every 8 nt, although within certain genomic regions, this can be considerably less often. Some reports suggest that the NGG PAM sequence can be replaced by NAG [25], but the relative efficiencies of cleavage have not been directly tested. Since sgRNAs therefore only have a 20 nt (target) + 2 nt (PAM) specificity determinant, and recent studies have shown that mismatches can be tolerated within the target sequence [25–30], targets must be carefully chosen to minimise the potential for off target effects. Ideally, sgRNA sequences should be chosen whose closest off target site differs by at least 4 nt, but this requirement can be relaxed if the mutations cluster towards the 30 end of the sgRNA, closest to the PAM. Mutations of only 1–3 nt within the final 10 nt of the target sequence often prevent cleavage, especially if they are next to each other. Several websites have recently become available to enable simple design of sgRNA target sequences that minimise potential off targeting (for example http://crispr.mit.edu/ [29], http://www.flyrnai.org/crispr/ [10], http://tools.flycrispr.molbio.wisc.edu/targetFinder/ [6], http://www.e-crisp.org/ E-CRISP/). For mutation of protein coding genes, it is often desirable to choose target sequences that will result in failure to produce a functional protein. Target sites should be chosen that are within the coding sequence of the gene to induce frameshifts, at the translational start codon or at the splice acceptor or donor sites of a common exon. This is because only 2 of 3 indels will result in a frameshift, whereas removal of a splice site or start codon will prevent a functional protein being produced. The indels produced can also be used to remove other functional sites within the genome such as transcription factor binding sites, miRNA target sites, splice sites and transcriptional start sites as well as mutating protein coding genes. There is considerable variability in the efficiency of different sgRNAs [6–12] (Table 1), so it is wise to design around three for each desired target. The reasons for this are not well understood,

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Section

2.3 2.4 2.5

2.6

2.5

sgRNA

Design of sgRNAs PCR of sgRNA template In vitro transcription of sgRNA and Cas9 mRNA Purification of sgRNA and Cas9 mRNA Embryo injection Growth to adulthood Backcrossing of mosaic adults Screening of adults for mutation by HRMA Growth of putative mutants to adulthood Screening of adult wings by HRMA + sequencing Backcrossing of adults Generation of stable stock

Cas9 mRNA

m7G

AAAAAAAAAAA

2d

14 d

14 d

~1 month

Normalised Fluorescence

2.2

A A AU GA A U G C CA G A A G GU AG U AU AA GC UA G A CG UA CG UA U AG UA AU UA C U GC CG CG UA GGNNNNNNNNNNNNNNNNNNNNG U AC AG CG UU AUC AAUGGC UUUU

1.0 0.8 Mutant WT

0.6 0.4 0.2 0.0 78

79

80

81 82 83 84 Temperature °C

85

86

87

mut T T T T C C T A A G A A AGCG G AA T T G A GCG wt T T T T C C T A A G C A C A C G A T C T C A T A C G

Fig. 1. Generation of targeted mutations by CRISPR/Cas9. Overview and timeline of mutant generation by CRISPR/Cas9 injection to Drosophila embryos. Numbers refer to sections within the text describing the processes.

but may be due to the energy change resulting from binding to the DNA, secondary structure formation in the RNA or accessibility of the underlying DNA sequence within chromatin. It is possible to test the efficacy of sgRNA cleavage more rapidly by HRMA analysis of DNA extracted from embryos 24 h after injection [6] or in cell lines [12]. 2.2.2. Design of sgRNA oligonucleotides Template for sgRNA transcription is made by PCR with a targetspecific oligonucleotide (CRISPR F) containing the T7 polymerase binding site, target sequence and a region complementary to a common reverse primer (CRISPR sgR) containing the rest of the sgRNA sequence (Fig. 2B). Positive controls targeting the yellow or white genes can also be included (Supplementary Table 2): 1. Add the following sequences to the 20 nt target sequence (in bold) to generate the CRISPR F oligonucleotide. If the target sequence begins with 1 or 2 G nucleotides, these can be substituted for the G nucleotide(s) immediately adjacent to the target sequence: GAAATTAATACGACTCACTATAGG NNNNNNNNNNNNNNNNNNNN GTTTAGAGCTAGAAATAGC T7 promoter

Target sequence

sgRNA backbone

2. Transcription from the T7 promoter begins with the GG adjacent to the target sequence, thereby extending it by 2 nt. If possible, it is optimal if the first two bases of the target sequence were GG, making the overall length 2 nt shorter. However, recent reports have suggested that addition of 2 bases to the 50 end of the sgRNA has relatively little impact on its efficacy [31]. 2.3. RNA production and purification 2.3.1. Production of sgRNA template 1. Set up PCR reactions using Phusion polymerase in a 100 ll total volume (20 ll 5 Phusion HF buffer, 67 ll ddH2O, 2 ll 10 mM dNTPs, 5 ll 10 lM CRISPR F primer, 5 ll 10 lM CRISPR sgR primer and 1 ll Phusion DNA polymerase). 2. Cycle samples under the following conditions: 98 °C 30 s, 35 cycles of [98°C 10 s, 60°C 30 s and 72°C 15 s, 72 °C 10 min, 4 °C hold. 3. Analyse 5 ll PCR product on a 2% agarose gel for purity and integrity of the approximately 100 nt product (Fig. 3A).

4. Purify the remaining 95 ll sample using a PCR purification kit (Qiagen), following the manufacturer’s instructions and eluting in 30 ll EB. It is also possible to use gel extraction at this stage if non-specific products are present. 5. Quantify concentration using a Nanodrop spectrophotometer. The expected yield should be around 4.5 lg at around 150 ng/ ll concentration. 2.3.2. In vitro transcription of sgRNA sgRNAs are generated by in vitro transcription of the sgRNA PCR template using the T7 MEGAscript kit (Ambion). Other in vitro transcription systems using T7 RNA polymerase can also be used. When producing and handling RNA, it is important to wear gloves, and clean equipment and benches with detergent prior to use to avoid RNAse contamination. Pipette tips with filters can also be beneficial to prevent contamination from pipettes. 1. Assemble a 20 ll reaction at room temperature to avoid precipitation of template DNA adding components in the indicated order (6 ll ddH2O, 2 ll ATP, 2 ll CTP, 2 ll GTP, 2 ll UTP, 2 ll 10 reaction buffer, 2 ll (300 ng) PCR product (from step 2.3.1), 2 ll enzyme mix containing T7 RNA polymerase and RNAse inhibitor). 2. Incubate at 37 °C for 4 h. 3. Add 1 ll turbo DNAse, mix and incubate for a further 15 min at 37 °C. 4. Stop the reaction by adding 115 ll ddH2O and 15 ll ammonium acetate stop solution. 5. Extract proteins by adding 150 ll phenol:chloroform:isoamyl alcohol (25:24:1) at pH 6.7 (Sigma), and vortex thoroughly for 30 s. 6. Separate phases by centrifugation at 10,000g for 3 min at room temperature, and remove the upper phase to a fresh tube. 7. Precipitate the RNA by addition of an equal volume (150 ll) of isopropanol. 8. Mix thoroughly, and incubate at 20 °C for greater than 15 min (can be left overnight). 9. Collect RNA by centrifugation at 17,000g for 15–30 min at 4 °C. 10. Wash pellet twice in 0.5 ml room temperature 70% ethanol, centrifuging at 17,000g for 3 min at 4 °C between each wash. 11. Remove the remaining liquid and dry RNA pellet for 3 min at room temperature.

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A

A A AU GA A U G C CA G A A G GU AG U AU AA GC UA G A CG A A U CG UA U G UA AU UA C U GC CG CG UA N N N N N N N N N N N N N N N N N N N N G U A C A G C G U U A U C A A U G G C U U U U -3’

Cas9

5’-

sgRNA

NNNNNNNNNNNNNNNNNNNN N N N N N N 3’-NNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNN-5’ Cleavage site NNNNNNNNNNNNNNNNNNNNNNNNNNN-3’ 5’-NNNNNNNNNN G N G N N PAM N NNNNNNNNNNNNNNNNNNNN

Target site

B

Target site

PAM

5’-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNN-3’ 3’-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNN-5’

Genomic DNA

1. Selection of target site T7 promoter

Target site

sgRNA

5’-GAAATTAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3’

CRISPR F 3’-CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA-5’

CRISPR sgR 2. Synthesis of CRISPR F

T7 promoter

Target site

sgRNA

5’-GAAATTAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3’ 3’-CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA-5’

3. PCR to generate template T7 promoter

Target site

sgRNA

5’-GAAATTAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’ 3’-CTTTAATTATGCTGAGTGATATCCNNNNNNNNNNNNNNNNNNNNCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA-5’

A A AU GA A U G C CA G A A G GU AG U AU AA GC UA G A CG A UA CG UA U G UA AU UA C U GC CG CG UA GGNNNNNNNNNNNNNNNNNNNNG U AC AG CG UU AUC AAUGGC UUUU

4. In vitro transcription

Fig. 2. Synthetic guide RNA (sgRNA) template design and production. (A) Schematic of Cas9-sgRNA complex showing target site (orange), protospacer adjacent motif (red) and cleavage sites (black triangles) in the DNA. Cas9 protein is indicated by a blue oval, and sgRNA sequence is shown, including the variable target region (orange). (B) Production of template DNA for in vitro transcription of sgRNA. DNA target site is indicated in orange, and PAM sequence in red. CRISPR F primer containing the T7 promoter (blue), target site (orange) and overlap with CRISPR sgR primer (purple) is shown. PCR is used to generate the template for in vitro transcription of the sgRNA. T7 polymerase transcription start site is indicated by an arrow, and mature RNA sequence is shown.

12. Resuspend in 30 ll RNAse-free ddH2O, measure concentration on a Nanodrop spectrophotometer, and dilute to 1 lg/ll. 2.3.3. Preparation of Cas9 template DNA Cas9 mRNA is generated by in vitro transcription of a linearised MLM3613 (Addgene plasmid 42251) plasmid template [5]: 1. Digest MLM3613 Cas9 vector with Pme I to linearize in a 50 ll reaction (10 lg MLM3613 plasmid, 5 ll 1 mg/ml BSA, 5 ll 10 NEBuffer 4, 5 ll (50 U) Pme I (NEB), made up to 50 ll with ddH2O). 2. Incubate at 37 °C for a minimum of 2 h (can be incubated overnight). 3. Stop linearization by adding 1/20 volume 0.5 M EDTA (2.5 ll) and 1/10 volume 3 M sodium acetate pH 5.2 (5 ll), and mix thoroughly. 4. Precipitate DNA by adding 2 volumes ethanol (115 ll) and incubating at 20 °C for at least 15 min.

5. Collect DNA at 17,000g for 15–30 min at 4 °C. 6. Remove residual fluid with a small pipette tip, and dry for 5 min at room temperature. 7. Resuspend pellet in 12 ll ddH2O, measure concentration on a Nanodrop spectrophotometer and dilute to 500 ng/ll. 2.3.4. In vitro transcription of Cas9 mRNA The mMessagemMachine T7 kit (Ambion) is used to perform in vitro transcription with T7 RNA polymerase in the presence of a 50 cap analog, followed by in vitro polyadenylation with the polyA tailing kit (Ambion) to mimic the structure of a mRNA: 1. Assemble a 20 ll reaction at room temperature to avoid precipitation of template DNA adding components in the indicated order (4 ll ddH2O, 2 ll (1 lg) linearized plasmid DNA, 10 ll 2 NTP/CAP mixture, 2 ll 10 reaction buffer and 2 ll enzyme mix). 2. Mix and incubate at 37 °C for 2 h.

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A

B DNA sgRNA

M

Cas9

6000 nt 4000 nt

5

6. Analyse 1 ll on a 1.2% agarose gel alongside an RNA ladder (Riboruler, Thermo scientific) to check the integrity of the RNA (Fig. 3C), and 1 ll on a Nanodrop spectrophotometer, to obtain a the RNA concentration. 7. Freeze at 80 °C until ready to inject.

500 bp 2.4. Embryo injection

200 nt

C 6000 nt 4000 nt

M

Cas9 / sgRNA

DNA 1500 bp 1000 bp 500 bp

200 nt

Fig. 3. Production of mRNA and sgRNA for injection. (A) Expected results of sgRNA PCR analysed on a 1% agarose gel. DNA marker (DNA) and two sgRNA PCR reactions are shown. (B) Expected result of Cas9 mRNA in vitro transcription analysed on a 1.2% agarose gel after polyadenylation. RNA marker (M) and sizes (nt) are indicated. (C) Purified RNA after mixing and purification of Cas9 mRNA and sgRNA analysed on a 1.2% agarose gel. RNA (M) and DNA (DNA) markers are indicated with sizes.

3. Add the following components to polyadenylate the transcript (36 ll ddH2O, 20 ll 5 E-PAP buffer, 10 ll 25 mM MnCl2, 10 ll 10 mM ATP, 4 ll E-PAP). Remove 2.5 ll before and after incubation to analyse efficient polyadenylation on an agarose gel. 4. Mix and incubate at 37 °C for 30 min. 5. Purify RNA with a RNeasy mini kit (Qiagen) using the following adapted protocol. 6. Add 350 ll buffer RLT and mix (do not add bmercaptoethanol). 7. Add 250 ll 100% ethanol and mix by pipetting. 8. Transfer sample to RNeasy mini spin column. 9. Centrifuge for 15 s at 10,000g at room temperature, and discard flow-through. 10. Add 500 ll RPE buffer and centrifuge for 15 s at 10,000g at room temperature, and discard flow-through. 11. Add 500 ll RPE buffer and centrifuge for 30 s at 10,000g at room temperature. 12. Transfer column to fresh 2 ml tube, and centrifuge for 2 min at 10,000g to dry column. 13. Transfer column to 1.5 ml collection tube, add 50 ll RNAse free water (prewarmed to 37 °C), incubate for 1 min, then centrifuge for 1 min at 10,000g at room temperature. 14. Measure concentration on a Nanodrop spectrophotometer, and freeze 10 lg aliquots at 80 °C (Fig. 3B). 2.3.5. Purification of Cas9–sgRNA mixture for injection Cas9 mRNA and sgRNA are mixed in an approximately 1:2 molar ratio, and further purified and concentrated before injection: 1. Mix 10 lg Cas9 mRNA and 0.5 lg sgRNA (20:1 (w/w) ratio), make up to 30 ll with ddH2O, add 3 ll 3 M sodium acetate, pH 5.2 and mix thoroughly. 2. Precipitate RNA mixture with 90 ll ethanol, and incubate at 20 °C for a minimum of 15 min. 3. Collect RNA by centrifugation at 17,000g for 30 min at 4 °C. 4. Wash twice in 100 ll room temperature 70% ethanol, centrifuging at 17,000g for 3 min at 4 °C between each wash. 5. Remove the remaining liquid, and dry at room temperature for 3 min before resuspending in 12 ll RNAse-free ddH2O.

Syncytial blastoderm stage embryos of any genotype are injected with the Cas9 mRNA and sgRNA mixture. Positive controls targeting the yellow or white genes provide a simple readout of efficient mutagenesis in the injected generation and should result in high efficiencies of mutagenesis (in our experiments yellow: 88% mosaic G0, 34.5% total mutant offspring; white: 25% mosaic G0, 10.2% total mutant offspring) (Fig. 4A–C, Supplementary Table 2). Here, we describe a protocol for injection through the chorion and vitelline membrane of the embryos, but it is also possible to dechorionate and dessicate the embryos before injection. 2.4.1. Preparation of embryos for injection 1. Flies of the desired genotype are expanded such that there are 5–10 bottles (6 oz) of 3–5 day old flies using standard techniques. 2. Flies are transferred to two small laying cages (5 cm diameter, 10 cm long; approximately 3–5 bottles of flies per cage). 3. A small amount (3–4 mm diameter pellet) of dried yeast slurry in water is placed onto 35 mm diameter apple juice agar plates. 4. Flies are incubated at 25 °C on a 12 h light/dark cycle, and plates changed every 3–4 h for at least 1 day prior to collection for injection to remove any embryos stored in the females, that would be of the wrong age. 5. On the day of injection, plates are swapped hourly for 2–3 h prior to collection. 6. Collect embryos for injection for 30 min at 25 °C. Do not increase the time, or embryos may have begun cellularisation, and injection will not reach the germline. 7. Ideally, subsequent steps should be carried out at 18 °C or room temperature to slow development of the embryos. 8. Wash embryos off the plate using a paintbrush and water into a collection basket, and rinse thoroughly to remove any yeast. 9. Blot embryos to remove excess water, and transfer to a droplet of water on an 18 mm square coverslip put on a microscope slide. 10. Line up the embryos at the edge of the coverslip, so that they are touching each other, and their posterior end is towards the edge of the coverslip. The anterior end is marked by the dorsal appendages. 11. Continue until 50–100 embryos are lined up. Note the number of embryos, and remove any that have obviously aged too long. 12. Add water to allow the embryos to slide on the coverslip. 13. Leave embryos to dry for a few minutes. This will cause them to adhere to the surface of the glass. Check that they do not move by using a dry paintbrush. 14. Cover the embryos in injection oil (a 1:1 mixture of halocarbon oils 700 and 27 (Sigma)), and leave for a few minutes to penetrate between the chorion and vitelline membrane. This makes it easier to validate correct injection. 2.4.2. Injection of embryos 1. Prepare the injection needle by back-filling an Eppendorf Femtotip II with 3 ll RNA mixture (from step 2.3.5).

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1.0

Normalised Fluorescence

A

Normalised Fluorescence

6

0.8 0.6

white mosaics Non-mosaics

0.4 0.2 0.0

0.8 0.6

yellow mosaics Non-mosaics

0.4 0.2 0.0

86

87

88

89 90 91 Temperature °C

92

93

94

82

0.4

83

84

85 86 87 Temperature °C

88

89

88

89

90

91

0.4

0.3

Δ Fluorescence

Δ Fluorescence

1.0

white mosaics Non-mosaics

0.2 0.1

0.3

yellow mosaics Non-mosaics

0.2 0.1 0.0

0.0 86

87

88

89

90

91

92

93

94

Temperature °C

82

83

84

85

86

87

90

91

Temperature °C

B

C CCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGATGCCCTGCGGCGATCGAAAGGCAA CCATTGAGCAGTCGCATTCCGGATGGCGATACTTGGATGCCCaGCGGCGATCGAAAGGCAA CCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGAT-----GCGGCGATCGAAAGGCAA CCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGA------GCGGCGATCGAAAGGCAA CCATTGAGCAGTCGCAT--------------------------GCGGCGATCGAAAGGCAA CCATTGAGCAGTCGCATCC-----------------------TGCaGCGATCGAAAGGCAA CCATTGAGCA------------------------------CCTGCGGCGATCGAAAGGCAA

AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACTGGAACCGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACAC------CGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACTGG------GGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACT--------GGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACT--------GGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTT------------CCGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGT--------------CCGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCG----------------------ACCGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGCC-----------------------GTGGGCATCGGCAATACC AGTGGATGAGTGTGGT--------------------------------CATCGGCAATACC AGTGGATGAGTGTG-----------------------------GTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGG----------------------------------GCA-TACC -----------------------------------------CCGTGGGCATCGGCAATACC AGTGGATGAGTGTGGTCGGCTGTGGGTTTT---------------GGG-------------

Fig. 4. Expected results from HRMA and sequencing. (A) Results of HRMA analysis of mosaic (G0) flies injected with Cas9 and sgRNAs targeting the white (left panels) or yellow (right panels) genes. Wild type and non-mosaic flies are indicated in grey, and putative mosaic flies in red. Normalised fluorescent melt curves are shown in the upper panels, and difference curves relative to a wild type control in the lower panels. (B) Mosaic white (left panel) and yellow (right panel, dotted line) expression can be seen in the eyes and abdomen of injected (G0) flies. (C) Sequencing of PCR products spanning the sgRNA target site shows the expected indel mutations. Target site is indicated in orange, PAM sequence in red and cleavage site by a black triangle.

2. Make sure the needle is mounted at an approximately 30° angle relative to the microscope slide. 3. Mount the needle in a micromanipulator (Eppendorf Transferman MK2), and break gently against the side of a coverslip stuck onto a microscope slide with a small drop of water whilst expelling liquid. This is best done under a 20 objective to allow more delicate manipulations. Store under injection oil until ready to use. 4. The best needles are those that are chamfered at the end, to allow better penetration through the membrane, and approximately 1.0–1.5 lm outer diameter.

5. Adjust injection pressure (Pi) to around 1000–1200 hPa (Eppendorf Femtojet Express), and compensation pressure (Pc) to around 50–100 hPa, such that a 0.1 s pulse gives a droplet of around a quarter the size of an embryo. 6. Do not keep RNA in the needle at room temperature for more than 1 h to avoid degradation. 7. Place the coverslip with the aligned embryos at around 45° to the slide, so that embryos can be injected at this angle. This avoids the thickest part of the chorion, and reduces chances of losing the germ cells from the posterior end of the embryo.

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Fig. 5. Crossing scheme. All offspring from the CRISPR injected G0 generation are individually crossed to a marker/balancer line (marker/balancer, upper panel). After 1 week injected flies are removed, DNA extracted, and HRMA used to identify mosaic animals (upper left panel). Offspring generated from those crosses set up with mosaic flies are analysed for mutations by HRMA and sequence analysis of DNA isolated from a single wing (middle panel). Offspring harbouring indels of interest are crossed to a balancer line, and used to make stable lines (lower right panel). Mutations are confirmed by sequencing of heterozygotes (lower left panel) or homozygotes.

8. Inject embryos off centre into the posterior end, making sure the needle penetrates both the chorion and vitelline membrane, but does not penetrate more than one third of the length of the embryo. 9. Upon injection, a clearing should be visible in the embryo, indicating that RNA has been injected. Break the end off the needle on the coverslip if it gets clogged. 10. We note that it is also possible to use a simplified microinjection system, using a 50 ml syringe to provide the pressure necessary for injection. 2.4.3. Growth of injected embryos 1. After injection, remove the coverslip from the slide, and drain away the excess oil by embedding it vertically in an apple juice agar plate for 5–10 min. 2. Take each coverslip, and transfer to a food tube with the embryo side downwards, nearly in contact with the food. A small amount of yeast paste will encourage hatched larvae to move away from the coverslip. 3. Place in a humid chamber, made by putting damp tissue paper in a beaker, and covering in cling film, and incubate at 25 °C for 48 h. 4. Count the number of hatched embryos, remove coverslips, and incubate at 25 °C for the rest of development. We note that using this technique, survival rates of embryos to adulthood are typically less 10–40%, even with control injections. 2.5. Generation of stable mutant lines Mutant flies are identified by HRMA of mosaic flies in the injected (G0) generation, and of single wings in the subsequent gen-

erations. The nature of the mutation can be determined in heterozygotes by sequencing of single wings before setting up crosses to make stocks. Stable fly lines are made by crossing to appropriate balancer chromosomes, depending on the genomic location of the induced mutation (Fig. 5).

2.5.1. Crossing scheme 1. Upon injection, each potentially mosaic fly is set up in an individual cross with two flies from a marked balancer line for the desired chromosome (e.g. Sco/CyO). 2. Flies are removed after 7 day or once the cross has been established, and analysed for the presence of mosaic mutations by high resolution melt analysis (HRMA). 3. Those crosses that are potentially mosaic are maintained, and the others discarded. 4. Offspring should contain heterozygous mutants if they have been transmitted through the germline. They are analysed for the presence of the mutation by extracting DNA from a single wing, and performing HRMA (see Section 2.6). 5. Sequencing of the PCR product from the HRMA analysis can also be performed to identify the nature and extent of the mutation before setting up crosses. 6. Whilst analysis is being performed, flies can be isolated in 1.5 ml tubes containing 0.3 ml fly food, and small holes in the lid to allow air circulation. 7. Mutants of interest are then crossed individually to the marked balancer line as before, and offspring used to set up stable lines. 8. Mutations are verified by HRMA and sequencing of PCR products from these lines.

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2.6. High resolution melt analysis Several techniques for detecting indel mutations have been successfully used, including Cel-I (Surveyor) and T7 endonuclease I assays that enzymatically cleave heteroduplexes, or loss of restriction enzyme sites within the sgRNA target site. Whilst these techniques can be used to detect and follow indel mutations, we have found that HRMA is the simplest, quickest and most sensitive system, especially when detecting mutations in mosaic animals from the injected generation. However, it does require dedicated HRMA equipment, or a quantitative PCR machine with appropriate analysis software. HRMA is used to identify mutations in mosaic animals and follow mutations in subsequent generations by analysis of single wings. It can also be used to rapidly screen for off target mutagenesis in offspring to identify and remove individuals that may contain such mutations. 2.6.1. DNA preparation from whole flies 1. Place one fly in a 0.2 ml tube and mash the fly for 10 s with a pipette tip containing 50 ll of squishing buffer (10 mM Tris– HCl, pH 8.2, 1 mM EDTA, 25 mM NaCl, 200 lg/ml proteinase K) without expelling any liquid (sufficient liquid escapes from the tip). Then expel the remaining buffer. 2. Incubate at 37 °C for 30 min then inactivate the proteinase K by heating to 95 °C for 2 min. 2.6.2. DNA preparation from single wings 1. PCR from single wings is adapted from Carvalho et al. [32]. Remove one wing from a fly near to the base, and transfer to a 0.2 ml tube. 2. Carefully cover the wing with 10 ll of wing buffer (10 mM Tris– HCl, pH 8.2, 1 mM EDTA, 25 mM NaCl, 400 lg/ml proteinase K), ensuring that it is submerged. 3. Incubate at 37 °C for 60 min then inactivate the proteinase K by heating to 95 °C for 2 min. 2.6.3. High resolution melt analysis High resolution melt analysis uses PCR in the presence of a saturating fluorescent DNA dye (LC Green). Indel mutations result in a change in melting profile of the product, which can be detected by analysis of the loss of fluorescent signal with increasing temperature: 1. Design primers to amplify 100–200 bp product spanning the sgRNA target site using standard techniques (e.g. primerBLAST – http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Small products increase the sensitivity of the approach. Note that the LC Green increases apparent Tm by 1–3 °C. 2. Annealing temperatures should be optimised for each primer pair to give a single product, with no primer dimer formation. Analyse products on a 2% agarose gel. If there are problems with non-specific products, primer dimers or low product yield, DMSO can be added to 5% final concentration. 3. It is important to include appropriate controls, including DNA from a non-injected fly, a reaction with no template, and to use an amplicon at a non-targeted locus. 4. Set up PCR reactions with genomic DNA (5 ll 2 Hotshot Diamond PCR mix, 0.2 ll 10 lM forward primer, 0.2 ll 10 lM reverse primer, 1 ll 10 LC Green, 1 ll DNA (from step 2.5.1 or 2.5.2) and 2.6 ll ddH2O). Use black plates with white wells (Clent Life science), and optically transparent seals (Life technologies). 5. Amplify on the following thermal cycle: 95 °C 5 min, 45 cycles of [95 °C 10 s, 55–65 °C 30 s and 72 °C 30 s], 95 °C 30 s, 25 °C 30 s, 4 °C hold.

6. Perform high resolution melt analysis from 70–98 °C, and use appropriate software to normalise fluorescent intensity and temperature shift the melting curves. We use the LightScanner and CallIT software (Idaho Technologies), but most quantitative PCR machines provide this facility. Expected results from mosaic and heterozygous animals are shown in Figs. 4A and 5. 7. The control injections targeting yellow and white can also be tested by HRMA using the primers in Supplementary Table 2 (yHMA, w2HMA). Detectable changes in the melt curve should be expected in approximately 88% of yellow and 25% of white flies in the injected (G0) generation. 2.6.4. Sequencing of PCR products The PCR products from HRMA analysis can also be used to obtain sequence information across the cleavage site to determine the extent and nature of the mutation. Note that this is only really possible from heterozygote or homozygote flies, not mosaic flies from the injected generation, since they will only contain a relatively small proportion of mutant tissue: 1. PCR products after HRMA analysis are treated with Exo I and shrimp alkaline phosphatase to degrade primers and remove dNTPs from the reaction (5 ll PCR product and 2 ll Exo-SAP IT). 2. Incubate at 37 °C for 15 min and inactivate the enzyme at 80 °C for a further 15 min. 3. Sanger sequencing can be performed directly on this sample with one of the primers used for amplification. 4. Although heterozygous mutations will result in overlapping peaks in the sequencing trace at the cleavage site, it is possible to identify the sequence of the other allele because we know the wild type sequence of the PCR product. It is therefore possible to manually read the sequence from the overlapping peaks present in the sequencing trace (Fig. 5). 3. Troubleshooting 1. No cleavage is detectable after HRMA (a) Some sgRNAs do not work well – Test other sgRNA sequences in the same target gene. (b) Efficiency of cleavage is too low to detect – Try analysing flies from the subsequent generation by wing PCR and HRMA. (c) Mutations are lethal during development – It may be that highly efficient sgRNAs make sufficient homozygous mutant tissue to prevent development. Try reducing RNA concentration that is injected, and analysing hatching and survival rates. (d) Injections have not worked – Test with known positive control such as yellow or white (Fig. 4, Supplementary Table 2). (e) Sequence is wrong – Reorder oligonucleotide. (f) Cas9 mRNA or sgRNA degraded – Run stock solutions of RNA on an agarose gel to check integrity (Fig. 3B and C), and repeat the in vitro transcription if necessary. 2. RNA production (a) No or low yield of RNA from Cas9 mRNA transcription reaction – Repurify DNA template, ensure reagents are fresh and reaction is assembled at room temperature. (b) Degraded RNA – Ensure solutions are RNAse free and gloves are worn. 3. Embryo injection

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(a) Poor survival or fertility – If this is sgRNA-dependent, dilute the RNA to a lower concentration and re-inject. Repurify RNA to remove contaminants. Ensure oil is aerated by vortexing before use, and drained thoroughly after injection. Reduce size of needle to minimise damage to embryos. Inject smaller volumes of liquid by reducing injection pressure or time. Make sure that humidity levels are high after injection. (b) Needle clogs regularly – Break more off the tip of the needle, and select needles with chamfered ends.

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ported by the UK Medical Research Council and the European Research Council (DARCGENs, project number 249869). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ymeth.2014.02. 019. References

4. HRMA detection (a) No amplification or multiple products – Optimise annealing temperature and try adding 5% DMSO to the reaction. Redesign primers. (b) Double peak in melting analysis – Optimise amplification as above, redesign primers. Analyse product on a gel. Single products can sometimes give two phase melting curves, which may still be useful to detect indels. (c) Variability in negative controls – Variability is often higher in wing PCRs, due to low concentration of DNA. Try using two wings, or if possible whole flies. Make DNA preps immediately before analysis. Optimise amplification as above or redesign primers. 4. Concluding remarks/perspective Despite the high density of transposable element mutagenesis in Drosophila, approximately 40% of annotated genes still lack a mutagenic insertion [33,34]. Many of these have no known function, despite having orthologs in other organisms. The use of the CRISPR/Cas9 system to create novel mutant alleles in essentially any gene will therefore allow investigation of the function of this interesting subset of genes refractory to current mutagenesis techniques. It will also allow analysis of other small functional sites within the genome, such as splice sites, promoters and transcription factor binding sites to investigate their function. The ability to generate mutations in any genetic background will also enable investigation of double or triple mutants that may be tightly linked genetically, and allow studies of redundancy or genetic interactions between such genes. We have recently demonstrated that it is possible to generate mutations in two genes in a single step by injection of two sgRNAs at the same time (unpublished observations), as has been demonstrated in other systems [3]. It will also make analysis of certain behavioural or other phenotypes that are highly dependent on genetic background simpler and easier. In the future, it will no doubt be possible to combine this technique with homologous targeting [12,35,36] to allow defined deletions and insertions and enable controlled genetic changes to be made. These techniques will add tremendously to our ability to manipulate the genome in a targeted manner. Combined with the powerful developmental genetic systems already available, this will allow Drosophila to remain at the forefront of genetic analysis for many years to come. Acknowledgements Further information concerning experimental methods and links to discussion groups and other information are provided at the OxfCRISPR website (http://oxfcrispr.org). The high resolution fly images in Fig. 4B were kindly provided by Nicolas Gompel. The authors would like to thank Professor Chris Ponting for his support, and Dr. Charlotte Tibbit and Dr. Sarah Cooper for critical reading of the manuscript. The CRISPR/Cas9 projects were sup-

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Cas9 mediated genome engineering in Drosophila.

Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect nov...
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