crispr/cas9 mediated genome engineering in drosophila

Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Methods

journal homepage: www.elsevier .com/locate /ymeth

CRISPR/Cas9 mediated genome engineering in Drosophila

http://dx.doi.org/10.1016/j.ymeth.2014.02.0191046-2023/� 2014 Published by Elsevier Inc.

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).

Please cite this article in press as: A. Bassett, J.-L. Liu, Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.019

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 a b s t r a c t

Article history:Available online xxxx

Keywords:Drosophila melanogasterCRISPRCas9Genome engineeringTargeted mutagenesisRNA injection

Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simpleand efficient technique to generate and detect novel mutations in desired target genes in Drosophila mel-anogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encod-ing the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. Thesmall insertion and deletion mutations that result from inefficient non-homologous end joining at thissite are detected by high resolution melt analysis of whole flies and individual wings, allowing stablelines to be made within 1 month.

� 2014 Published by Elsevier Inc.

1. Introduction The DSBs produced can be repaired by non-homologous end

Our ability to design DNA binding factors with exquisite speci-ficity for desired target sequences has heralded a new wave of gen-ome engineering techniques that allow targeted modifications ofthe genome to be achieved in many organisms [1–14]. This newgenome engineering technology will enable more directed andelegant experiments to be performed to analyse structural andfunctional aspects of the genome.

The CRISPR/Cas9 system was discovered as a bacterial defencesystem against invading viral pathogens, which uses fragments ofRNA from the virus to target cleavage of the viral DNA throughcomplementary base pairing [15–20]. This system has recentlybeen shown to be active in other systems, including mammals[1–3], insects [6–12] and plants [13], and can be easily modifiedto target double strand breaks (DSB) at any desired target sequenceby supplying it with a short guide RNA that is complementary tothe target site within the DNA. The endogenous system involvesthree components. The Cas9 protein is an endonuclease that bindsto a structure within a trans-acting CRISPR RNA (tracrRNA). ThetracrRNA base pairs with a CRISPR RNA (crRNA), the first 20 nt ofwhich determine the specificity of the Cas9 endonuclease. A sim-plified two component system has been described that fuses thetracrRNA and crRNA into a single synthetic guide RNA (sgRNA),making delivery of the components easier [1,2,18].

joining (NHEJ) or homologous recombination (HR), and both canbe useful to introduce mutations into the underlying DNA [21].NHEJ repair is error prone, and often results in small insertionsor deletions (indels) at the cut site, that can be mutagenic. Target-ing two DSBs can also result in the deletion of intervening se-quences, to generate longer deficiencies [6]. Induction of a DSBalso enhances rates of HR repair, which can be used to enhancegene 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 theCas9 and sgRNA components have been developed [6–12] (Table 1).The Cas9 protein can be introduced by injection of mRNA or anexpression vector into the early embryo [6–8], or by using atransgenic strain that produces the Cas9 protein under a germ-line-specific or ubiquitous promoter [9–11]. The sgRNA itself canbe produced by in vitro transcription [7,8], or expressed from apol III promoter derived from the U6 snRNA gene [6,9–11]. Theuse of a pol III promoter avoids capping and polyadenylation ofthe transcript, which may inhibit its activity. Again, in vitrotranscribed sgRNA or an expression plasmid can be injected intoDrosophila embryos, or transgenic strains can be produced thatexpress the sgRNA ubiquitously. These techniques can be used indifferent combinations, and each has advantages in certain circum-stances, or for specific experiments (Table 1). For instance, the high-est reproducibility and efficiency of mutagenesis can be achieved bycrossing two transgenic lines, but it relies on generating a transgenicline expressing each desired sgRNA, which is relatively timeconsuming. Although giving good mutagenesis efficiency, all ofthe techniques involving transgenic Cas9 expression rely oninjection or crossing to the transgenic fly lines, making it difficultto compound mutations with pre-existing alleles, or inject into

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Table 1Comparison 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 hsp70 T7 Sp6 nos vasa nosCas9 delivery DNA

injectionmRNAinjection

mRNA injection Transgenic Transgenic Transgenic

sgRNA promoter U6 T7 T7 U6 U6 U6a, U6b, nos-mini

sgRNA delivery DNAinjection

sgRNAinjection

sgRNA injection Transgenic DNAinjection

DNA injection

Target genes Yellow Yellow, white Yellow, K81, CG3708, CG9652, kl-3,light, RpL15

White, neuropeptide genes(Ast, capa, Ccap, Crz, Eh,Mip, npf),mir-219, mir-315

EGFP, mRFP White

Mosaic G0 (%)a 6–66 4–88 35.7–80 N/A N/A N/AGermline mutants

(among fertile flies) (%)b5.9–20.7 0–79 35.7–100 0–100 35–71 0–100

F1 mutant overall (%)c 0.25–1.37 0–34.5 2.1–98.9 0–99.4 7.7–24.7 0–74.2Overall Timescaled �1 month �1 month �1 month �2–3 months �1 month �1 monthApplicable to all genetic

backgroundseYes Yes Yes No No 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.

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

different genetic backgrounds. The described technique has theadvantage that it can be performed in essentially any geneticbackground, and there is no possibility of integration of DNAconstructs into the genome, but does require care in the productionand handling of the injected RNA.

Here we describe a detailed methodology to produce and injectmRNA encoding the Cas9 protein, and in vitro transcribed sgRNAsthat can result in high efficiencies of mutagenesis of desired targetgenes by inefficient NHEJ. Up to 88% of flies have mosaic mutationsin the target gene, which can be transmitted to up to 34.5% of totalF1 offspring [7] (Table 1). We also describe the application of highresolution melt analysis (HRMA) to provide a simple and effectivesystem of detection of the resulting indel mutations to enablegeneration of stable mutant lines [7]. This technique utilises thefact that indel mutations change the melting temperature of PCRproducts spanning the target site to rapidly and accurately detectmosaic and heterozygous mutant flies.

2. Materials and methods

2.1. Overview

sgRNAs are designed to target the gene of interest that mini-mise potential off target effects and maximise mutagenic effi-ciency, and templates for their transcription are generated by asimple PCR. The sgRNA and mRNA encoding the Cas9 protein aregenerated by in vitro transcription, purified and coinjected intoDrosophila embryos of essentially any genotype. Mosaic flies areidentified by HRMA, and heterozygous mutant offspring from theseflies are selected by analysis of PCR products from single wings byHRMA and sequencing. These flies are used to make stable stocksthat can be used for further analysis. An overview of the processwith approximate timings is shown in Fig. 1, and reagents requiredare listed in Supplementary Table 1.

2.2. sgRNA design

2.2.1. Target site choiceCas9 is guided to 20 nt target sequences in the genome that are

complementary to the 50 end of the sgRNA, and these sequences

Please cite this article in press as: A. Bassett, J.-L. Liu, Methods (2014), http://d

must be followed by an NGG protospacer adjacent motif (PAM) se-quence (Fig. 2A). The PAM sequence does not appear in the sgRNA,but is nevertheless required by the Cas9 protein for efficient endo-nucleolytic cleavage of the DNA. These sequences can be on eitherstrand of the DNA, making the expected frequency of such sitesapproximately every 8 nt, although within certain genomic re-gions, this can be considerably less often. Some reports suggestthat the NGG PAM sequence can be replaced by NAG [25], butthe 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 mis-matches can be tolerated within the target sequence [25–30], tar-gets must be carefully chosen to minimise the potential for offtarget effects. Ideally, sgRNA sequences should be chosen whoseclosest off target site differs by at least 4 nt, but this requirementcan be relaxed if the mutations cluster towards the 30 end of thesgRNA, closest to the PAM. Mutations of only 1–3 nt within the fi-nal 10 nt of the target sequence often prevent cleavage, especiallyif they are next to each other. Several websites have recently be-come available to enable simple design of sgRNA target sequencesthat minimise potential off targeting (for example http://crispr.mi-t.edu/ [29], http://www.flyrnai.org/crispr/ [10], http://tools.fly-crispr.molbio.wisc.edu/targetFinder/ [6], http://www.e-crisp.org/E-CRISP/).

For mutation of protein coding genes, it is often desirable tochoose target sequences that will result in failure to produce afunctional protein. Target sites should be chosen that are withinthe coding sequence of the gene to induce frameshifts, at the trans-lational start codon or at the splice acceptor or donor sites of acommon exon. This is because only 2 of 3 indels will result in aframeshift, whereas removal of a splice site or start codon will pre-vent a functional protein being produced. The indels produced canalso be used to remove other functional sites within the genomesuch as transcription factor binding sites, miRNA target sites, splicesites and transcriptional start sites as well as mutating protein cod-ing genes.

There is considerable variability in the efficiency of differentsgRNAs [6–12] (Table 1), so it is wise to design around three foreach desired target. The reasons for this are not well understood,

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http://crispr.mit.edu/

http://crispr.mit.edu/

http://www.flyrnai.org/crispr/

http://tools.flycrispr.molbio.wisc.edu/targetFinder/

http://tools.flycrispr.molbio.wisc.edu/targetFinder/

http://www.e-crisp.org/E-CRISP/

http://www.e-crisp.org/E-CRISP/


Design of sgRNAsPCR of sgRNA template

In vitro transcription of sgRNA and Cas9 mRNAPurification of sgRNA and Cas9 mRNA

Embryo injection

Backcrossing of mosaic adultsScreening of adults for mutation by HRMA

Growth to adulthood

Backcrossing of adults

Growth of putative mutants to adulthoodScreening of adult wings by HRMA + sequencing

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

A. Bassett, J.-L. Liu / Methods xxx (2014) xxx–xxx 3

but may be due to the energy change resulting from binding to theDNA, secondary structure formation in the RNA or accessibility ofthe underlying DNA sequence within chromatin. It is possible totest the efficacy of sgRNA cleavage more rapidly by HRMA analysisof DNA extracted from embryos 24 h after injection [6] or in celllines [12].

2.2.2. Design of sgRNA oligonucleotidesTemplate for sgRNA transcription is made by PCR with a target-

specific oligonucleotide (CRISPR F) containing the T7 polymerasebinding site, target sequence and a region complementary to acommon reverse primer (CRISPR sgR) containing the rest of thesgRNA sequence (Fig. 2B). Positive controls targeting the yellowor white genes can also be included (Supplementary Table 2):

1. Add the following sequences to the 20 nt target sequence (inbold) to generate the CRISPR F oligonucleotide. If the targetsequence begins with 1 or 2 G nucleotides, these can be substi-tuted for the G nucleotide(s) immediately adjacent to the targetsequence:

GAAATTAATACGACTCACTATAGGT7 promoter

NNNNNNNNNNNNNNNNNNNNTarget sequence

GTTTAGAGCTAGAAATAGCsgRNA backbone

2. Transcription from the T7 promoter begins with the GG adjacentto the target sequence, thereby extending it by 2 nt. If possible, itis optimal if the first two bases of the target sequence were GG,making the overall length 2 nt shorter. However, recent reportshave suggested that addition of 2 bases to the 50 end of the sgRNAhas 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 totalvolume (20 ll 5� Phusion HF buffer, 67 ll ddH2O, 2 ll 10 mMdNTPs, 5 ll 10 lM CRISPR F primer, 5 ll 10 lM CRISPR sgR pri-mer and 1 ll Phusion DNA polymerase).

2. Cycle samples under the following conditions: 98 �C 30 s, 35cycles 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 andintegrity 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 elutingin 30 ll EB. It is also possible to use gel extraction at this stage ifnon-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 sgRNAsgRNAs are generated by in vitro transcription of the sgRNA PCR

template using the T7 MEGAscript kit (Ambion). Other in vitrotranscription 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 toavoid RNAse contamination. Pipette tips with filters can also bebeneficial to prevent contamination from pipettes.

1. Assemble a 20 ll reaction at room temperature to avoid pre-cipitation of template DNA adding components in the indi-cated order (6 ll ddH2O, 2 ll ATP, 2 ll CTP, 2 ll GTP, 2 llUTP, 2 ll 10� reaction buffer, 2 ll (300 ng) PCR product(from step 2.3.1), 2 ll enzyme mix containing T7 RNA poly-merase 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 ammo-

nium acetate stop solution.5. Extract proteins by adding 150 ll phenol:chloroform:iso-

amyl alcohol (25:24:1) at pH 6.7 (Sigma), and vortex thor-oughly for 30 s.

6. Separate phases by centrifugation at 10,000�g for 3 min atroom 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 than15 min (can be left overnight).

9. Collect RNA by centrifugation at 17,000�g for 15–30 min at4 �C.

10. Wash pellet twice in 0.5 ml room temperature 70% ethanol,centrifuging at 17,000�g for 3 min at 4 �C between eachwash.

11. Remove the remaining liquid and dry RNA pellet for 3 min atroom temperature.

x.doi.org/10.1016/j.ymeth.2014.02.019


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T7 promoter sgRNATarget site

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


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

5’-GAAATTAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3’

3’-CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA-5’


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Genomic DNA

1. Selection of target site

2. Synthesis of CRISPR F

4. In vitro transcription

3. PCR to generate template

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 polymerasetranscription start site is indicated by an arrow, and mature RNA sequence is shown.


12. Resuspend in 30 ll RNAse-free ddH2O, measure concentra-tion on a Nanodrop spectrophotometer, and dilute to 1 lg/ll.

2.3.3. Preparation of Cas9 template DNACas9 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 llreaction (10 lg MLM3613 plasmid, 5 ll 1 mg/ml BSA, 5 ll10� NEBuffer 4, 5 ll (50 U) Pme I (NEB), made up to 50 ll withddH2O).

2. Incubate at 37 �C for a minimum of 2 h (can be incubatedovernight).

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 mixthoroughly.

4. Precipitate DNA by adding 2 volumes ethanol (115 ll) and incu-bating at �20 �C for at least 15 min.


5. Collect DNA at 17,000�g 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 mRNAThe mMessagemMachine T7 kit (Ambion) is used to perform

in vitro transcription with T7 RNA polymerase in the presence ofa 50 cap analog, followed by in vitro polyadenylation withthe polyA tailing kit (Ambion) to mimic the structure of amRNA:

1. Assemble a 20 ll reaction at room temperature to avoid precip-itation of template DNA adding components in the indicatedorder (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.

x.doi.org/10.1016/j.ymeth.2014.02.019


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6000 nt4000 nt

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Fig. 3. Production of mRNA and sgRNA for injection. (A) Expected results of sgRNAPCR analysed on a 1% agarose gel. DNA marker (DNA) and two sgRNA PCR reactionsare shown. (B) Expected result of Cas9 mRNA in vitro transcription analysed on a1.2% 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 ona 1.2% agarose gel. RNA (M) and DNA (DNA) markers are indicated with sizes.


3. Add the following components to polyadenylate the tran-script (36 ll ddH2O, 20 ll 5� E-PAP buffer, 10 ll 25 mMMnCl2, 10 ll 10 mM ATP, 4 ll E-PAP). Remove 2.5 ll beforeand after incubation to analyse efficient polyadenylationon an agarose gel.

4. Mix and incubate at 37 �C for 30 min.5. Purify RNA with a RNeasy mini kit (Qiagen) using the follow-

ing adapted protocol.6. Add 350 ll buffer RLT and mix (do not add b-

mercaptoethanol).7. Add 250 ll 100% ethanol and mix by pipetting.8. Transfer sample to RNeasy mini spin column.9. Centrifuge for 15 s at 10,000�g 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, thencentrifuge 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 injectionCas9 mRNA and sgRNA are mixed in an approximately 1:2 mo-

lar 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 17,000�g for 30 min at 4 �C.4. Wash twice in 100 ll room temperature 70% ethanol, centrifug-

ing 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.


6. Analyse 1 ll on a 1.2% agarose gel alongside an RNA ladder(Riboruler, Thermo scientific) to check the integrity of theRNA (Fig. 3C), and 1 ll on a Nanodrop spectrophotometer, toobtain a the RNA concentration.

7. Freeze at �80 �C until ready to inject.

2.4. Embryo injection

Syncytial blastoderm stage embryos of any genotype are injectedwith the Cas9 mRNA and sgRNA mixture. Positive controls targetingthe yellow or white genes provide a simple readout of efficient muta-genesis in the injected generation and should result in high efficien-cies of mutagenesis (in our experiments yellow: 88% mosaic G0,34.5% total mutant offspring; white: 25% mosaic G0, 10.2% total mu-tant offspring) (Fig. 4A–C, Supplementary Table 2). Here, we de-scribe a protocol for injection through the chorion and vitellinemembrane of the embryos, but it is also possible to dechorionateand dessicate the embryos before injection.

2.4.1. Preparation of embryos for injection

1. Flies of the desired genotype are expanded such that thereare 5–10 bottles (6 oz) of 3–5 day old flies using standardtechniques.

2. Flies are transferred to two small laying cages (�5 cm diame-ter,�10 cm long; approximately 3–5 bottles of flies per cage).

3. A small amount (3–4 mm diameter pellet) of dried yeastslurry in water is placed onto 35 mm diameter apple juiceagar plates.

4. Flies are incubated at 25 �C on a 12 h light/dark cycle, andplates changed every 3–4 h for at least 1 day prior to collec-tion for injection to remove any embryos stored in thefemales, that would be of the wrong age.

5. On the day of injection, plates are swapped hourly for 2–3 hprior to collection.

6. Collect embryos for injection for 30 min at 25 �C. Do notincrease the time, or embryos may have begun cellularisa-tion, and injection will not reach the germline.

7. Ideally, subsequent steps should be carried out at 18 �C orroom temperature to slow development of the embryos.

8. Wash embryos off the plate using a paintbrush and waterinto a collection basket, and rinse thoroughly to removeany yeast.

9. Blot embryos to remove excess water, and transfer to a drop-let of water on an 18 mm square coverslip put on a micro-scope slide.

10. Line up the embryos at the edge of the coverslip, so that theyare touching each other, and their posterior end is towardsthe edge of the coverslip. The anterior end is marked bythe dorsal appendages.

11. Continue until 50–100 embryos are lined up. Note the numberof 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 theydo not move by using a dry paintbrush.

14. Cover the embryos in injection oil (a 1:1 mixture of halocar-bon oils 700 and 27 (Sigma)), and leave for a few minutes topenetrate between the chorion and vitelline membrane. Thismakes it easier to validate correct injection.

2.4.2. Injection of embryos

1. Prepare the injection needle by back-filling an EppendorfFemtotip II with 3 ll RNA mixture (from step 2.3.5).

x.doi.org/10.1016/j.ymeth.2014.02.019


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0.3

AGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACTGGAACCGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACAC------CGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACTGG------GGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACT--------GGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTTTGGACACT--------GGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTT------------CCGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGT--------------CCGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCG----------------------ACCGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGCC-----------------------GTGGGCATCGGCAATACCAGTGGATGAGTGTGGT--------------------------------CATCGGCAATACCAGTGGATGAGTGTG-----------------------------GTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGG----------------------------------GCA-TACC-----------------------------------------CCGTGGGCATCGGCAATACCAGTGGATGAGTGTGGTCGGCTGTGGGTTTT---------------GGG-------------

CCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGATGCCCTGCGGCGATCGAAAGGCAACCATTGAGCAGTCGCATTCCGGATGGCGATACTTGGATGCCCaGCGGCGATCGAAAGGCAACCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGAT-----GCGGCGATCGAAAGGCAACCATTGAGCAGTCGCATCCCGGATGGCGATACTTGGA------GCGGCGATCGAAAGGCAACCATTGAGCAGTCGCAT--------------------------GCGGCGATCGAAAGGCAACCATTGAGCAGTCGCATCC-----------------------TGCaGCGATCGAAAGGCAACCATTGAGCA------------------------------CCTGCGGCGATCGAAAGGCAA

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 eyesand 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, PAMsequence 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 Transf-erman MK2), and break gently against the side of a coverslipstuck onto a microscope slide with a small drop of waterwhilst expelling liquid. This is best done under a 20�objective to allow more delicate manipulations. Store underinjection oil until ready to use.

4. The best needles are those that are chamfered at the end, toallow better penetration through the membrane, andapproximately 1.0–1.5 lm outer diameter.


5. Adjust injection pressure (Pi) to around 1000–1200 hPa(Eppendorf Femtojet Express), and compensation pres-sure (Pc) to around 50–100 hPa, such that a 0.1 s pulsegives a droplet of around a quarter the size of anembryo.

6. Do not keep RNA in the needle at room temperature formore than 1 h to avoid degradation.

7. Place the coverslip with the aligned embryos at around 45� tothe slide, so that embryos can be injected at this angle. Thisavoids the thickest part of the chorion, and reduces chancesof losing the germ cells from the posterior end of the embryo.

x.doi.org/10.1016/j.ymeth.2014.02.019


INJECTION

Mosaic +

X or MarkerBalancer

1 2 :

tnatuMBalancer X

1 2 :

tnatuMBalancer

HRMA (fly)

HRMA (wing)

HRMA and Sequencing

or

Nor

mal

ised

Flu

ores

cenc

e

1.0

0.8

0.6

0.0

0.2

0.4

Temperature °C48 58 6887 97 08 18 28 38 87

MutantWT

Nor

mal

ised

Flu

ores

cenc

e

1.0

0.8

0.6

0.0

0.2

0.4

Temperature °C68 8884787674 08 2827

MutantWT

C C C C CCT A A A A A TTT T GTmut

wt

sgRNAAAAAAAAAAAAm7GCas9 mRNA

T CA T CA GC CT A ATT T GT A AAG

GCGG AA T TGAGCG

MarkerBalancer

Seq

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 weekinjected 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 areanalysed 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 balancerline, 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 surethe needle penetrates both the chorion and vitelline mem-brane, but does not penetrate more than one third of thelength of the embryo.

9. Upon injection, a clearing should be visible in the embryo,indicating that RNA has been injected. Break the end offthe needle on the coverslip if it gets clogged.

10. We note that it is also possible to use a simplified microin-jection system, using a 50 ml syringe to provide the pressurenecessary for injection.

2.4.3. Growth of injected embryos

1. After injection, remove the coverslip from the slide, and drainaway the excess oil by embedding it vertically in an apple juiceagar plate for 5–10 min.

2. Take each coverslip, and transfer to a food tube with theembryo side downwards, nearly in contact with the food. Asmall amount of yeast paste will encourage hatched larvae tomove away from the coverslip.

3. Place in a humid chamber, made by putting damp tissue paper ina beaker, and covering in cling film, and incubate at 25 �C for 48 h.

4. Count the number of hatched embryos, remove coverslips, andincubate at 25 �C for the rest of development. We note thatusing this technique, survival rates of embryos to adulthoodare 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 in-jected (G0) generation, and of single wings in the subsequent gen-


erations. The nature of the mutation can be determined inheterozygotes by sequencing of single wings before setting upcrosses to make stocks. Stable fly lines are made by crossing toappropriate balancer chromosomes, depending on the genomiclocation of the induced mutation (Fig. 5).

2.5.1. Crossing scheme

1. Upon injection, each potentially mosaic fly is set up in an indi-vidual cross with two flies from a marked balancer line for thedesired chromosome (e.g. Sco/CyO).

2. Flies are removed after 7 day or once the cross has been estab-lished, and analysed for the presence of mosaic mutations byhigh resolution melt analysis (HRMA).

3. Those crosses that are potentially mosaic are maintained, andthe others discarded.

4. Offspring should contain heterozygous mutants if they havebeen transmitted through the germline. They are analysed forthe presence of the mutation by extracting DNA from a singlewing, and performing HRMA (see Section 2.6).

5. Sequencing of the PCR product from the HRMA analysis can alsobe performed to identify the nature and extent of the mutationbefore setting up crosses.

6. Whilst analysis is being performed, flies can be isolated in1.5 ml tubes containing 0.3 ml fly food, and small holes in thelid to allow air circulation.

7. Mutants of interest are then crossed individually to the markedbalancer line as before, and offspring used to set up stable lines.

8. Mutations are verified by HRMA and sequencing of PCRproducts from these lines.

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2.6. High resolution melt analysis

Several techniques for detecting indel mutations have been suc-cessfully used, including Cel-I (Surveyor) and T7 endonuclease I as-says that enzymatically cleave heteroduplexes, or loss ofrestriction enzyme sites within the sgRNA target site. Whilst thesetechniques can be used to detect and follow indel mutations, wehave found that HRMA is the simplest, quickest and most sensitivesystem, especially when detecting mutations in mosaic animalsfrom the injected generation. However, it does require dedicatedHRMA equipment, or a quantitative PCR machine with appropriateanalysis software. HRMA is used to identify mutations in mosaicanimals and follow mutations in subsequent generations by analy-sis of single wings. It can also be used to rapidly screen for offtarget mutagenesis in offspring to identify and remove individualsthat 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 apipette tip containing 50 ll of squishing buffer (10 mM Tris–HCl, pH 8.2, 1 mM EDTA, 25 mM NaCl, 200 lg/ml proteinaseK) without expelling any liquid (sufficient liquid escapes fromthe tip). Then expel the remaining buffer.

2. Incubate at 37 �C for 30 min then inactivate the proteinase K byheating 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 toa 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 byheating to 95 �C for 2 min.

2.6.3. High resolution melt analysisHigh resolution melt analysis uses PCR in the presence of a saturat-

ing fluorescent DNA dye (LC Green). Indel mutations result in achange in melting profile of the product, which can be detected byanalysis of the loss of fluorescent signal with increasing temperature:

1. Design primers to amplify 100–200 bp product spanning thesgRNA target site using standard techniques (e.g. primerBLAST– http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Smallproducts increase the sensitivity of the approach. Note thatthe LC Green increases apparent Tm by 1–3 �C.

2. Annealing temperatures should be optimised for each primerpair to give a single product, with no primer dimer formation.Analyse products on a 2% agarose gel. If there are problems withnon-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 DNAfrom a non-injected fly, a reaction with no template, and touse an amplicon at a non-targeted locus.

4. Set up PCR reactions with genomic DNA (5 ll 2� Hotshot Dia-mond PCR mix, 0.2 ll 10 lM forward primer, 0.2 ll 10 lMreverse primer, 1 ll 10� LC Green, 1 ll DNA (from step 2.5.1 or2.5.2) and 2.6 ll ddH2O). Use black plates with white wells (ClentLife science), and optically transparent seals (Life technologies).

5. Amplify on the following thermal cycle: 95 �C 5 min, 45 cyclesof [95 �C 10 s, 55–65 �C 30 s and 72 �C 30 s], 95 �C 30 s, 25 �C30 s, 4 �C hold.


6. Perform high resolution melt analysis from 70–98 �C, and useappropriate software to normalise fluorescent intensity andtemperature shift the melting curves. We use the LightScannerand CallIT software (Idaho Technologies), but most quantitativePCR machines provide this facility. Expected results frommosaic and heterozygous animals are shown in Figs. 4A and 5.

7. The control injections targeting yellow and white can also betested by HRMA using the primers in Supplementary Table 2(yHMA, w2HMA). Detectable changes in the melt curve shouldbe expected in approximately 88% of yellow and 25% of whiteflies in the injected (G0) generation.

2.6.4. Sequencing of PCR productsThe PCR products from HRMA analysis can also be used to ob-

tain sequence information across the cleavage site to determinethe extent and nature of the mutation. Note that this is only reallypossible from heterozygote or homozygote flies, not mosaic fliesfrom the injected generation, since they will only contain a rela-tively small proportion of mutant tissue:

1. PCR products after HRMA analysis are treated with Exo I andshrimp alkaline phosphatase to degrade primers and removedNTPs 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 �Cfor a further 15 min.

3. Sanger sequencing can be performed directly on this samplewith one of the primers used for amplification.

4. Although heterozygous mutations will result in overlappingpeaks in the sequencing trace at the cleavage site, it is possibleto identify the sequence of the other allele because we know thewild type sequence of the PCR product. It is therefore possibleto manually read the sequence from the overlapping peakspresent in the sequencing trace (Fig. 5).

3. Troubleshooting

1. No cleavage is detectable after HRMA

(a) Some sgRNAs do not work well – Test other sgRNAsequences in the same target gene.

(b) Efficiency of cleavage is too low to detect – Try analysingflies from the subsequent generation by wing PCR andHRMA.

(c) Mutations are lethal during development – It may be thathighly efficient sgRNAs make sufficient homozygous mutanttissue to prevent development. Try reducing RNA concentra-tion that is injected, and analysing hatching and survivalrates.

(d) Injections have not worked – Test with known positive con-trol 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), andrepeat the in vitro transcription if necessary.

2. RNA production

(a) No or low yield of RNA from Cas9 mRNA transcription reac-tion – Repurify DNA template, ensure reagents are fresh andreaction is assembled at room temperature.

(b) Degraded RNA – Ensure solutions are RNAse free and glovesare worn.

3. Embryo injection

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http://www.ncbi.nlm.nih.gov/tools/primer-blast/



(a) Poor survival or fertility – If this is sgRNA-dependent, dilutethe RNA to a lower concentration and re-inject. RepurifyRNA to remove contaminants. Ensure oil is aerated by vor-texing before use, and drained thoroughly after injection.Reduce size of needle to minimise damage to embryos. Injectsmaller volumes of liquid by reducing injection pressure ortime. 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.

4. HRMA detection

(a) No amplification or multiple products – Optimise annealingtemperature and try adding 5% DMSO to the reaction. Rede-sign primers.

(b) Double peak in melting analysis – Optimise amplification asabove, redesign primers. Analyse product on a gel. Singleproducts can sometimes give two phase melting curves,which may still be useful to detect indels.

(c) Variability in negative controls – Variability is often higherin wing PCRs, due to low concentration of DNA. Try usingtwo wings, or if possible whole flies. Make DNA preps imme-diately before analysis. Optimise amplification as above orredesign primers.

4. Concluding remarks/perspective

Despite the high density of transposable element mutagenesisin Drosophila, approximately 40% of annotated genes still lack amutagenic insertion [33,34]. Many of these have no known func-tion, despite having orthologs in other organisms. The use of theCRISPR/Cas9 system to create novel mutant alleles in essentiallyany gene will therefore allow investigation of the function of thisinteresting subset of genes refractory to current mutagenesis tech-niques. It will also allow analysis of other small functional siteswithin the genome, such as splice sites, promoters and transcrip-tion factor binding sites to investigate their function.

The ability to generate mutations in any genetic backgroundwill also enable investigation of double or triple mutants thatmay be tightly linked genetically, and allow studies of redundancyor genetic interactions between such genes. We have recentlydemonstrated that it is possible to generate mutations in twogenes in a single step by injection of two sgRNAs at the same time(unpublished observations), as has been demonstrated in othersystems [3]. It will also make analysis of certain behavioural orother phenotypes that are highly dependent on genetic back-ground simpler and easier.

In the future, it will no doubt be possible to combine this tech-nique with homologous targeting [12,35,36] to allow defined dele-tions and insertions and enable controlled genetic changes to bemade. These techniques will add tremendously to our ability tomanipulate the genome in a targeted manner. Combined withthe powerful developmental genetic systems already available, thiswill allow Drosophila to remain at the forefront of genetic analysisfor many years to come.

Acknowledgements

Further information concerning experimental methods andlinks to discussion groups and other information are provided atthe OxfCRISPR website (http://oxfcrispr.org). The high resolutionfly images in Fig. 4B were kindly provided by Nicolas Gompel.The authors would like to thank Professor Chris Ponting for hissupport, and Dr. Charlotte Tibbit and Dr. Sarah Cooper for criticalreading of the manuscript. The CRISPR/Cas9 projects were sup-


ported by the UK Medical Research Council and the European Re-search Council (DARCGENs, project number 249869).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ymeth.2014.02.019.

References

[1] L. Cong, F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P.D. Hsu, X. Wu, W. Jiang,L.A. Marraffini, F. Zhang, Science 339 (2013) 819–823.

[2] P. Mali, L. Yang, K.M. Esvelt, J. Aach, M. Guell, J.E. DiCarlo, J.E. Norville, G.M.Church, Science 339 (2013) 823–826.

[3] H. Wang, H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, R.Jaenisch, Cell 153 (2013) 910–918.

[4] M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, J. Doudna, Elife 2 (2013) e00471.[5] W.Y. Hwang, Y. Fu, D. Reyon, M.L. Maeder, S.Q. Tsai, J.D. Sander, R.T. Peterson,

J.R. Yeh, J.K. Joung, Nat. Biotechnol. 31 (2013) 227–229.[6] S.J. Gratz, A.M. Cummings, J.N. Nguyen, D.C. Hamm, L.K. Donohue, M.M.

Harrison, J. Wildonger, K.M. O’Connor-Giles, Genetics 194 (2013) 1029–1035.[7] A.R. Bassett, C. Tibbit, C.P. Ponting, J.L. Liu, Cell Rep. 4 (2013) 220–228.[8] Z. Yu, M. Ren, Z. Wang, B. Zhang, Y.S. Rong, R. Jiao, G. Gao, Genetics 195 (2013)

289–291.[9] S. Kondo, R. Ueda, Genetics 195 (2013) 715–721.

[10] X. Ren, J. Sun, B.E. Housden, Y. Hu, C. Roesel, S. Lin, L.P. Liu, Z. Yang, D. Mao, L.Sun, Q. Wu, J.Y. Ji, J. Xi, S.E. Mohr, J. Xu, N. Perrimon, J.Q. Ni, Proc. Natl. Acad. Sci.USA 110 (2013) 19012–19017.

[11] Z.L. Sebo, H.B. Lee, Y. Peng, Y. Guo, Fly (Austin) 8 (2013) 1–6.[12] A.R. Bassett, C. Tibbit, C.P. Ponting, J.-L. Liu, Biol. Open 3 (2014) 42–49.[13] Q. Shan, Y. Wang, J. Li, Y. Zhang, K. Chen, Z. Liang, K. Zhang, J. Liu, J.J. Xi, J.L. Qiu,

C. Gao, Nat. Biotechnol. 31 (2013) 686–688.[14] J.E. DiCarlo, J.E. Norville, P. Mali, X. Rios, J. Aach, G.M. Church, Nucleic Acids

Res. 41 (2013) 4336–4343.[15] R. Barrangou, C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D.A.

Romero, P. Horvath, Science 315 (2007) 1709–1712.[16] J.E. Garneau, M.E. Dupuis, M. Villion, D.A. Romero, R. Barrangou, P. Boyaval, C.

Fremaux, P. Horvath, A.H. Magadan, S. Moineau, Nature 468 (2010) 67–71.[17] G. Gasiunas, R. Barrangou, P. Horvath, V. Siksnys, Proc. Natl. Acad. Sci. USA 109

(2012) E2579–E2586.[18] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, E. Charpentier, Science

337 (2012) 816–821.[19] M. Ishino, Y. Sawada, T. Yaegashi, M. Demura, K. Fujinaga, Virology 156 (1987)

414–416.[20] S.J. Brouns, M.M. Jore, M. Lundgren, E.R. Westra, R.J. Slijkhuis, A.P. Snijders, M.J.

Dickman, K.S. Makarova, E.V. Koonin, J. van der Oost, Science 321 (2008) 960–964.

[21] M. Bibikova, M. Golic, K.G. Golic, D. Carroll, Genetics 161 (2002) 1169–1175.[22] A. Choulika, A. Perrin, B. Dujon, J.F. Nicolas, Mol. Cell. Biol. 15 (1995) 1968–

1973.[23] G.B. Gloor, N.A. Nassif, D.M. Johnson-Schlitz, C.R. Preston, W.R. Engels, Science

253 (1991) 1110–1117.[24] F. Smih, P. Rouet, P.J. Romanienko, M. Jasin, Nucleic Acids Res. 23 (1995) 5012–

5019.[25] P. Mali, J. Aach, P.B. Stranges, K.M. Esvelt, M. Moosburner, S. Kosuri, L. Yang,

G.M. Church, Nat. Biotechnol. 31 (2013) 833–838.[26] T.J. Cradick, E.J. Fine, C.J. Antico, G. Bao, Nucleic Acids Res. 41 (2013) 9584–

9592.[27] Y. Fu, J.A. Foden, C. Khayter, M.L. Maeder, D. Reyon, J.K. Joung, J.D. Sander, Nat.

Biotechnol. 31 (2013) 822–826.[28] F.A. Ran, P.D. Hsu, C.Y. Lin, J.S. Gootenberg, S. Konermann, A.E. Trevino, D.A.

Scott, A. Inoue, S. Matoba, Y. Zhang, F. Zhang, Cell 154 (2013) 1380–1389.[29] P.D. Hsu, D.A. Scott, J.A. Weinstein, F.A. Ran, S. Konermann, V. Agarwala, Y. Li,

E.J. Fine, X. Wu, O. Shalem, T.J. Cradick, L.A. Marraffini, G. Bao, F. Zhang, Nat.Biotechnol. 31 (2013) 827–832.

[30] S.W. Cho, S. Kim, Y. Kim, J. Kweon, H.S. Kim, S. Bae, J.-S. Kim, Genome Res.(2013) 132–141.

[31] L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin, W.A.Lim, Cell 152 (2013) 1173–1183.

[32] G.B. Carvalho, W.W. Ja, S. Benzer, Biotechniques 46 (2009) 312–314.[33] H.J. Bellen, R.W. Levis, Y. He, J.W. Carlson, M. Evans-Holm, E. Bae, J. Kim, A.

Metaxakis, C. Savakis, K.L. Schulze, R.A. Hoskins, A.C. Spradling, Genetics 188(2011) 731–743.

[34] H.J. Bellen, R.W. Levis, G. Liao, Y. He, J.W. Carlson, G. Tsang, M. Evans-Holm, P.R.Hiesinger, K.L. Schulze, G.M. Rubin, R.A. Hoskins, A.C. Spradling, Genetics 167(2004) 761–781.

[35] K.J. Beumer, J.K. Trautman, A. Bozas, J.L. Liu, J. Rutter, J.G. Gall, D. Carroll, Proc.Natl. Acad. Sci. USA 105 (2008) 19821–19826.

[36] K.J. Beumer, J.K. Trautman, K. Mukherjee, D. Carroll, G3 (Bethesda) 3 (2013)657–664.

[37] A.R. Bassett, J.-L. Liu, J. Genet. Genomics 41 (2014) 7–19.

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