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, CRISPR/Cas9 and Gal4/UAS Combination for Cell-Specific

, The CRISPR/Cas9 37 system represents a major step forward towards achieving precise and targeted gene 38 disruption. Being readily applicable for the creation of knockout loci in a great 39 variety of animal models used in neuroscience studies, this technology has led to 40 significant advances in the fields of developmental and functional neurobiology 41 (Heidenreich and Zhang 2016). Nonetheless, constitutive gene disruption is often 42 associated with side effects, such as compensation mechanisms and embryonic 43 lethality, representing an important limitation on the analysis of phenotypes specific 44 to the nervous system, since neural circuits are fully established at late stages of 45 development. Recently, studies in worms (Shen et al. 2014), fruit flies (Port et al. 46 2014), mice (Platt et al. 2014) and zebrafish (Ablain et al. 2015) have pioneered the 47 use of the CRISPR/Cas9 methodology to generate conditional gene knockouts via 48 tissue-specific expression of cas9, the analysis of gene function has relied on mutagenesis 36 approaches leading to the generation of loss-of-function alleles

, Importantly, one of the most common methodologies ensuring cell-specific expres-51 sion of transgenes in zebrafish is the Gal4-UAS binary system

. Davison, Gene-and enhancer-trap methods have been applied to 55 establish a significant number of Gal4 transgenic lines, 2007.

. Asakawa, Notably, in these lines the Gal4 open reading frame (ORF) is randomly 59 integrated in the fish genome through Tol2-based transposition, and the insertion 60 site is not mapped; therefore, the sequence of the promoter elements driving Gal4 61 expression is unknown, 2007.

, Our strategy does not require 65 previous knowledge of promoter sequences to induce cas9 expression since this is 66 provided by cell type-specific Gal4 transcription. Additionally, to enable the anal-67 ysis of the phenotypes arising from Cas9-induced gene disruption, we marked the 68 population of the cas9-expressing cells by using the viral T2A self-cleaving peptide 69 (Provost et al. 2007), ensuring the stoichiometric synthesis of the Cas9 enzyme and 70 the fluorescent reporter GFP from the same mRNA. To test our conditional knock-71 out strategy, we used our vector system to target the tyrosinase (tyr) locus, coding 72 for a key enzyme involved in melanin production (Camp and Lardelli, UAS-mediated expression of the Cas9 enzyme with a constitutive expression of 64 sgRNAs driven by PolIII U6 promoter sequences, 2001.

S. Albadri,

M. ;. Campbell and . Auer, To target the kif5aa gene with 123 the 2C-Cas9 system in single RGC, we used the Tg(isl2b:gal4) line. As expected, 124 after injection of our construct into one, 2013.

, UAS:brainbow) fish, we could observe a strong decrease in 126 total branch length in YFP-or Cerulean-expressing RGC (potentially kif5aa mutant) 127 compared to tdTomato-fluorescent RGC, Tg(isl2b:gal4) and Tg

, The use of the Gal4/UAS system allows the targeting 130 of a gene of choice in any cell population. The combination of this bipartite system 131 with simultaneous activation of Cas9 And Cre enzymes in progenitor or differen-132 tiated cells enables first, the genetic lineage tracing of mutant cells and second, the 133 detection of cell-autonomous gene inactivation at single cell resolution. Addition-134 ally, permanent labeling of knockout cells offers the possibility of investigating 135 gene function in adult animals, conclusion, the 2C-Cas9 system represents a versatile tool to induce biallelic 129 conditional gene inactivation

, Crispr/Cas9 application for the generation of loss-of-141 function alleles, RNA guide nucleases can be used for more sophisticated genome 142 modifications such as homologous recombination (HR) or non-homologous end 143 joining (NHEJ)-mediated knockin

. Davison, The possible integration of exogenous genes into any given loci 150 and the analysis of their function in the living animal have dramatically improved 151 over the past few years with the development of genome editing technologies. Prior 152 to this recent explosion in the field of knockin generation, conventional transgenic 153 zebrafish lines were generated by Tol2-mediated transgenesis, which has success-154 fully allowed the making of hundreds of new reporter lines essential to the study of 155 particular gene functions in vivo, Crispr/Cas9-Mediated Knockin Approaches in Zebrafish 147 With its advantage of transparency, the zebrafish model organism rapidly emerged 148 as a powerful experimental system for studies in genetics, developmental biology 149 and neurobiology, 2007.

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, To circumvent this limitation, we present here a simple and versatile protocol to achieve tissue-specific gene disruption based on the Cas9 expression under the control of the Gal4/upstream activating sequence binary system. In our method, we couple Cas9 with green fluorescent protein or Cre reporter gene expression. This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells, and to follow them in vivo via reporter gene expression. Importantly, because none of the tools that we present here are restricted to zebrafish, similar approaches are readily applicable in virtually any organism where transgenesis and DNA injection are feasible. other harbors U6 cassettes for the transcription of sgRNAs (U6:sgRNA). Therefore, double carriers would show tissue-specific phenotypes subsequent to heat-shock and Cre activation. expression, we use a viral T2A self-cleaving peptide followed by the GFP CDS enabling the synthesis of the fluorescent reporter from the same transcript (UAS: Cas9T2AGFP). Two U6 promoters are subcloned in the same construct to drive ubiquitous transcription of the sgRNAs. BsmbI and BsaI restriction sites allow the cloning of the 20-bp target sequence at the transcription start site, Abstract In the last few years the development of CRISPR/Cas 9-mediated genome editing techniques has allowed the efficient generation of loss-of-function alleles in several model organisms including zebrafish

, Step 2: cloning of the target sequences of the sgRNAs displaying the highest mutation rate at the TSS of the U6 promoters in the pUAS:Cas9T2AGFP plasmid (Fig. 1C and D)

, Technical procedure Step 1: Design of sgRNAs targeting the genomic locus of interest and assessment of their mutagenesis efficiency sgRNA design ? Select a target site for sgRNA synthesis by using one of the available online tools

, ? Filter the research for oligonucleotides suitable for cloning at the TSS of the T7 in the pDR274 vector (Addgene ref 42250)

, Run the digestion product on a 0.8% agarose gel and extract the linear plasmid with a gel and polymerase chain reaction (PCR) extraction kit

, ? Mix 3 mL of 100 mM forward and reverse oligonucleotides containing the target sequence and add 14 mL of TE buffer (10 mM Tris-Cl

, Heat the mixture 10 min at 95 C and cool down for 30 min at room temperature to allow the annealing of the oligonucleotides

, Schematic of the pUAS:Cas9T2AGFP;U6sgRNA1;U6sgRNA2 plasmid; (B) schematic of the pUAS:Cas9T2ACre;U6sgRNA1;U6sgRNA2 plasmid; (C) insertion of the first target sequence into the transgenesis vector at the BsaI cloning sites; (D) insertion of the second target sequence into the transgenesis vector at the BsmbI cloning sites; (E) schematic view of cross of zebrafish Gal4

. U6sgrna1, U6sgRNA2 plasmid; (F) schematic representation of cross of zebrafish Gal4 transgenic driver to line carrying a floxed reporter allele and injection of the pUAS: Cas9T2ACre;U6sgRNA1

, ? Transform the reaction in TOP10 competent cells (ThermoFisher Scientificd, pp.2800-2802

, Inoculate each colony into 2 ml of LB-ampicillin medium and incubate at 37 C overnight with agitation. ? Purify DNA using a miniprep kit ? Sequence the plasmid with T7 specific primer to verify the presence of the target sequence ? Repeat the entire process with BsmBI enzyme using oligonucleotides containing overhangs for BsmBI cloning, ? Plate on LB-ampicillin plates ? The next day, pick two colonies from the transformation plate

, Step 3: Establishing tissue-specific gene inactivation (Fig. 1E and 2) Microinjection of pUAS:CAS9T2AGFP;U6:sgRNA1;U6:sgRNA2 plasmid ? Choose a specific Gal4 driver line based on the population of cells where gene function needs to be analyzed

, Prepare the injection mix as described: 30 ng/mL of pUAS:CAS9T2AGFP;U6:sgRNA1;U6:sgRNA2 construct þ50 ng/mL of Tol2 mRNA þRNAse, DNAse-free water up to 5 mL ? Inject with pUAS:CAS9T2AGFP;U6:sgRNA1;U6:sgRNA2 into one-celle stage embryos from an incross of a Tg, ? Set up mating couples of the chosen Gal4 transgenic line. ?

, ? Screen embryos with GFP-positive cells in the expected Gal4 expression pattern

, ? Screen for Fo adults carrying the transgene in the germline and cross them with the chosen Gal4 transgenic line

. Rationale-for, . Design-of-a-vector, . System, . Clonal, . Of et al., SYSTEM promoter regulating Gal4 transcription guarantees synchronized translation of Cas9 and Cre enzymes from the same mRNA, ensuring targeted mutagenesis at the chosen genomic locus and expression of the floxed reporter within the same cell. The fluorescence of the reporter will be stable throughout the entire life of the fish and, most importantly, will be inherited in all the cells deriving from the same Cas9-expressing progenitor (Fig. 3). Our strategy, named 2C-Cas9 (Cre-mediated recombination for Clonal analysis of Cas9 mutant cells) offers the possibility to induce site-specific mutagenesis in a selected cell population and

, Schematic of tissue-specific gene disruption and permanent clonal labeling of Cas9 -expressing cells after injection of the pUAS:Cas9T2ACre;U6sgRNA1;U6sgRNA2 plasmid in a cross of a specific Gal4 driver to a floxed reporter line (2C-Cas9)

, ? Wash twice in PBS-Tw, 5 min each

, ? Incubate with 1 mg/mL collagenase diluted in PBS according to the stage (1dpf, day post fertilization: 10 min; 2 dpf: 20 min; 3 dpf: 75 min, vol.4

, ? Wash three times in PBS-Tw, 5 min each

, ? Incubate for 1 h at room temperature in blocking solution

, ? Wash three times in PBS-Tw, 5 min each

, ? Incubate overnight at 4 C with primary antibodies diluted in blocking solution at the appropriate concentrations

, ? Wash three times in PBS-Tw, 5 min each

, ? Incubate overnight at 4 C with secondary antibodies diluted in blocking solution at the appropriate concentrations

, ? Wash three times in PBS-Tw, 5 min each

, ? Incubate overnight in 30% sucrose; 0.02% sodium azide; PBS solution. ? Embed the embryos in Tissue-Tek O.C.T. compound after removal of the sucrose and place the resulting blocks on dry ice before sectioning

, Section the blocks with a thickness of 14 mm and mount the sections on Fisherbrand Superfrost plus slides

, ? Wash twice in PBS-Tw, 5 min each time

, ? Incubate for 1 h at room temperature in blocking solution (10% normal goat serum in PBS-Tw)

, ? Incubate overnight at 4 C with primary antibodies diluted in blocking solution. ? Wash three times in PBS-Tw

, ? Incubate for 2 h with secondary antibodies diluted in blocking solution. ? Wash five times in PBS-Tw, 5 min each

, Add Vectashield drops on the sections and place coverslips on the slides

, Molecular assessment of the mutagenesis efficiency via fluorescence-activated cell sorting and genome locus sequencing The molecular assessment of the mutagenesis efficiency of the 2C-Cas9 system in the selected Gal4 line requires the isolation of the fluorescent Cas9-expressing cells. Fluorescence-activated cell sorting (FACS) might be used to isolate the marked Cas9-expressing cells from the wild-type nonfluorescent cell population. After separation, total DNA can be extracted from the two pools of cells and the targeted locus can be

, ? Dissociate the embryos (n > 200) as previously described by Manoli & Driever, 2012.

, ? Perform cell sorting and collect cells in lysis buffer (E.G. NucleoSpin Tissue kit

. Machereyenageld740952, , vol.10

, ? Extract genomic DNA following kit instructions

, ? PCR amplify the targeted genomic loci. ? Clone PCR amplicons into the pCR-bluntII-TOPO vector. ? Isolate plasmid DNA from single colonies

, ? Sequence the plasmids and identify mutant alleles by comparison with the wildtype sequence

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