To generate gdf6b(lf) mutants, we used TALEN genome editing. TALEN’s were designed targeting exon 1 of gdf6b (TAL1 sequence: GTCAGCATCACTGTTAT ; TAL2 sequence: CCTTGATCGCCCTTCT ). TALENs were assembled using the Golden Gate TALEN kit (Addgene) per the manufacturer’s instructions. TALEN plasmids were linearized and transcribed with mMESSAGE mMACHINE kit (Ambion). Zebrafish embryos were injected with 50 pg of mRNA of each TALEN arm. Injected embryos (F0) were matured to breeding age and outcrossed. Resulting offspring (F1) were genotyped by extraction genomic DNA from fin clips per standard protocol and PCR amplification with gdf6b primers. F1 offspring carrying mutations by genotyping were sequenced to identify mutations predicted to lead to loss of function of gdf6b. Following identification of candidate zebrafish by sequencing, zebrafish were bred to generate homozygous gdf6b(lf) mutations. Whole RNA was isolated from homozygous gdf6b(lf) embryos at 20 HPF and qRT-PCR was used to determine effective depletion of gdf6b transcripts. Primers for genotyping and qRT-PCR are listed in the Key Reagents section.
Golden gate talen kit
Manufactured by Addgene
Sourced in United States
The Golden Gate TALEN kit provides the necessary components to assemble and express customizable transcription activator-like effector nucleases (TALENs) for genome editing applications. The kit includes plasmids encoding TALEN repeat variable di-residues, scaffold, and other supporting elements required for TALEN assembly and expression.
Automatically generated - may contain errors
Lab products found in correlation
Nuclease free water, by Promega (2 mentions)
Sp6 polymerase mmessage mmachine kit, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine kit, by Thermo Fisher Scientific (1 mentions)
Ptz57r t vector, by Thermo Fisher Scientific (1 mentions)
Pgem 5zf plasmid, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Mmessage mmachine t3 kit, by Thermo Fisher Scientific (1 mentions)
Hcas9 expression vector, by Addgene (1 mentions)
Plvx puro, by Takara Bio (1 mentions)
10 protocols using golden gate talen kit
1
Generating gdf6b Loss-of-Function Mutants
2
Generating Zebrafish esr1 Mutants
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To obtain the zebrafish mutant line, specific TALEN target sites were designed locating on the third exon (Figure 1A ) of zebrafish esr1 (Gene ID: 259252), which encodes the DNA-binding domain (Figure 1C ). Paired TALENs were constructed with the Golden Gate TALEN Kit (Addgene, Cambridge, MA, USA) as reported (34 (link), 35 (link)). Approximately 300 pg TALEN mRNAs were microinjected into one-cell stage zebrafish embryos and then reared at the temperature of 28.5°C. Twenty four hours after injection, 10 embryos were collected for DNA extraction to check whether the targeted genomic fragment was deleted. The target genomic regions were amplified by PCR and subcloned into the pTZ57R/T vector (Fermentas). Single colonies were genotyped by sequencing.
To obtain germline mutations, the P0 generation zebrafish were raised to adulthood and mated to wild-type fish to generate heterozygous F1 offspring. The heterozygous F1 generation fish were genotyped via fin clip assay and the individuals with frame-shift sequence alterations were selected. Males and females of F1generation carrying the same mutation were mated to produce F2 homozygous mutants which were genotyped via PCR and sequencing. Specific primers for PCR genotyping are listed in Supplementary Table1 .
To obtain germline mutations, the P0 generation zebrafish were raised to adulthood and mated to wild-type fish to generate heterozygous F1 offspring. The heterozygous F1 generation fish were genotyped via fin clip assay and the individuals with frame-shift sequence alterations were selected. Males and females of F1generation carrying the same mutation were mated to produce F2 homozygous mutants which were genotyped via PCR and sequencing. Specific primers for PCR genotyping are listed in Supplementary Table
3
TALEN and CRISPR/Cas9 Plasmid Design
4
TALEN-Mediated Zebrafish Genome Editing
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Paired TALENs were designed and assembled as described previously [47 (link)] using the Golden Gate TALEN Kit (Addgene, Cambridge, MA, USA). The DNA sequences that were targeted by each TALEN pair are shown in Figure 3 A. A native enzyme restriction site, PstI, in the spacer region between the two TALEN arms was used for nucleotide change detection. Next, mRNA was synthesized using the T3 mMESSAGE mMACHINE Kit (Ambion, Austin, TX, USA) using the backbone plasmids pCS2TAL3DD and pCS2TAL3RR. Approximately 200–500 pg of mRNA was microinjected into 1- to 2-cell-stage zebrafish embryos. Next, 10–20 embryos following their hatching were collected for genotyping, and the target region was amplified using the primers in Table 1 . In this assay, the remainder of the embryos was raised to adults and were mated with wild-type zebrafish to generate F1 offspring. The F1 adults were genotyped by enzyme digestion and PCR product sequencing. The F2 generation was obtained from F1 strains harboring the mutations.
5
TALE-Mediated Targeting of Huntingtin
6
TALEN and CRISPR Gene Editing Protocols
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TALEN-binding domains were designed and developed following published guidelines (Cermak et al., 2011 (link)). Each binding domain was created by two rounds of the ‘Golden Gate’ assembly method (Cermak et al., 2011 (link)), where each TAL module was linked together and cloned on a single plasmid backbone using type IIS restriction enzymes BsaI and Esp3I. Full-length constructs were subsequently cloned on either pcGoldy-TALEN (TALEN-I3.1, -I3.2, I3.3 and I4) or on pCS2TAL3-RRR/DDD (TALEN-I2) expression vectors for expression in human cells. The Golden Gate TALEN kit and expression vectors were obtained from Addgene (Cambridge, MA, USA).
CRISPRs were designed following the Church's protocol (Mali et al., 2013 (link)). The expression vectors for the gRNAs were generated by ordering a 455 bp DNA fragment (composed of U6 promoter, target sequence, guide RNA scaffold and termination signal) as gBlocks from IDT (Leuven, Belgium) and then cloning them in pUC19 plasmid. The hCas9 expression vector was instead obtained from Addgene.
CRISPRs were designed following the Church's protocol (Mali et al., 2013 (link)). The expression vectors for the gRNAs were generated by ordering a 455 bp DNA fragment (composed of U6 promoter, target sequence, guide RNA scaffold and termination signal) as gBlocks from IDT (Leuven, Belgium) and then cloning them in pUC19 plasmid. The hCas9 expression vector was instead obtained from Addgene.
7
Generation of Cyp21a2 Gene Mutant Zebrafish
8
TALEN Design and Assembly Protocol
9
Bicistronic CRISPR Tools for Gene Editing
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The BioID2 was amplified by PCR from the Myc-BioID2-MCS plasmid (Addgene #74,223, Watertown, MA), fused with human ANO5, mouse Ano6 or mouse Mg53 cDNA, and inserted into pLVX-puro (Clontech, San Jose, CA) to obtain pLVX-hANO5-BioID2, pLVX-mAno6-BioID2, and pLVX-BioID2-mMG53, respectively. BVES-myc, POPDC2-myc and POPDC3 were constructed by inserting the corresponding cDNA into pCDNA3.1-myc backbone. ANO5 related plasmids were described previously [39 (link)]. The truncation or deletion mutant BVES constructs were generated by overlapping PCR. The Ano5-targeting TALEN pairs were assembled using the Golden Gate TALEN kit (Addgene #1000000016) as previously described [25 (link)] and the TALEN pairs were coupled with 2A peptide [40 (link)]. The protein sequences of Ano5-TALENs are provided in the Additional file 1 : Fig. S6. pCMV-AncBE4max plasmid was obtained from Addgene (#112094). The pCMV-AncBE4-GFP was generated by ligation of SacI-EcoR1 fragment from pCMV-AncBE4max and the EcoRI/AgeI-digested GFP fragment into SacI/AgeI linearized pCMV-AncBE4max backbone. The annealed gRNA oligos (targeting mouse Bves) were cloned into pLenti-OgRNA-Zeo plasmid as previously described [41 (link), 42 (link)]. All plasmids used in this study are listed in Additional file 1 : Table S2.
10
Generation and Screening of cyp21a2 Mutants
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The pair of TALENs targeting exon 2 of the cyp21a2 gene was generated with the Golden Gate TALEN Kit (Addgene, Cambridge, MA). TALEN target sites were determined by TAL Effector Nucleotide Targeter Version 2.0 (21 (link)). The sequences of the repeat-variable di-residues (RVDs) in the TAL Effector DNA-binding domains were RVD TALE1 (left): NG-HD-NG-NH-NH-NG-HD-HD-NG-HD-NH-HD-NG-HD-NG-HD and RVD TALE2 (right): NG-NH-NH-NH-HD-NH-NI-NH-NI-NG-HD-HD-NI-NH-HD-NI-NG-NH-NG. TALEN mRNA was synthesized with an SP6 polymerase mMessage mMachine Kit (Life Technologies, Waltham, MA) after 1 µg of plasmid DNA was digested with NotI.
TALENs were injected into one-cell-stage embryos, with 1 nL of injection solution containing 50 or 150 ng/µL of each TALEN diluted in nuclease-free water (Promega) plus 0.1% phenol red. F0 generations were grown to adulthood from the injected embryos and screened for transmission of cyp21a2 mutations. Identified founders were outcrossed to fish to generate F1 generations. The F1 generation fish were screened for heterozygous cyp21a2 mutations. F1 fish with defined cyp21a2 mutant alleles were outcrossed to their respective genetic backgrounds to generate the F2 generation. The heterozygous mutant fish of the F2 generation were incrossed to study cyp21a2 mutant phenotypes in embryos and larvae.
TALENs were injected into one-cell-stage embryos, with 1 nL of injection solution containing 50 or 150 ng/µL of each TALEN diluted in nuclease-free water (Promega) plus 0.1% phenol red. F0 generations were grown to adulthood from the injected embryos and screened for transmission of cyp21a2 mutations. Identified founders were outcrossed to fish to generate F1 generations. The F1 generation fish were screened for heterozygous cyp21a2 mutations. F1 fish with defined cyp21a2 mutant alleles were outcrossed to their respective genetic backgrounds to generate the F2 generation. The heterozygous mutant fish of the F2 generation were incrossed to study cyp21a2 mutant phenotypes in embryos and larvae.
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