Cas9-green fluorescence protein (GFP) (cat no.10008100), crRNA, tracrRNA (cat no.1072533), and ATTO550-labeled tracrRNA (cat no. 1075928) were obtained from Integrated DNA Technologies Inc. (Coralville, IA, USA). crRNA was designed to target the tyrosinase gene of C57BL/6 mice (5′-GGGTGGATGACCGTGAGTCC-3′), which participates in melanin biosynthesis [10 (link)]. This gene is specifically expressed in retinal pigment epithelial cells of the eye, choroidal melanocytes, and hair follicle melanocytes in mammals [11 ]. It is possible to discriminate the results of genome editing from the eye color of offspring derived from C57BL/6 × ICR embryos without genetic analysis by knocking out the tyrosinase gene. The nuclease solution for embryo electroporation contained 200 ng/μl Cas9-GFP, 15 μM crRNA, 15 μM tracrRNA or a mixture solution with 7.5 μM tracrRNA and 7.5 μM tracrRNA-ATTO550 in Opti-MEM (Thermo Fisher Scientific Inc., MA, USA) [8 (link)] was prepared just before electroporation.
Tracrrna
Manufactured by Thermo Fisher Scientific
Sourced in United States
TracrRNA is a critical component of the CRISPR-Cas9 gene editing system. It functions as a guide RNA, forming a complex with the Cas9 enzyme to identify and bind to specific DNA sequences for targeted genome modification.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Neon transfection system mpk5000, by Thermo Fisher Scientific (1 mentions)
Cas9 protein, by Integrated DNA Technologies (1 mentions)
Crrna, by Integrated DNA Technologies (1 mentions)
Atto550 labeled tracrrna, by Integrated DNA Technologies (1 mentions)
Crrna, by Thermo Fisher Scientific (1 mentions)
Tracrrna, by Integrated DNA Technologies (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Geneart platinum cas9 nuclease, by Thermo Fisher Scientific (1 mentions)
Lipofectamine crisprmax cas9 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Bstbi, by New England Biolabs (1 mentions)
Truecut cas9 protein v2, by Thermo Fisher Scientific (1 mentions)
6 protocols using tracrrna
1
CRISPR-Cas9 Mediated Tyrosinase Knockout
2
FOXRED1 Knockout in HEK293T Cells
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HEK293T cells were electroporated (Neon Transfection System, MPK5000, ThermoFisher) with Alt-R Sp HiFi Cas9 nuclease v3, tracrRNA, and FOXRED1 crRNA (Hs.Cas9.FOXRED1.1.AA and Hs.Cas9.FOXRED1.1.AC), all from Integrated DNA Technologies (Coralville, IA) per the manufacturer’s instructions. Single clones were expanded and plated in both DMEM + 10% FBS media containing 0.4 mM uridine, 2 mM pyruvate and either 25 mM glucose or 10 mM galactose. Clones that failed to grow in galactose had genomic DNA Sanger sequenced for evidence of editing, and clones were subject to Western blot to confirm FOXRED1 deletion, and one of these that appeared to grow well was further studied.
3
CRISPR Deletion of Peptide Lv Gene
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To delete the nucleotide sequence encoding peptide Lv, we designed two guide RNAs (gRNAa, b) comprising 20-nucleotide sequences targeting exon 2 of the peptide Lv gene (5′-CTGTGGTGCTCTCAATGTCA-3′ and 5′-ACCTCTCATTTCCGTCGCCG-3′). The two gRNAs coupled with constant regions of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). Along with the gRNAs, Cas9 proteins (Integrated DNA Technologies, Coralville, IA, USA) were electroporated into fertilized C57BL/6J zygotes according to a previously published protocol (19 (link)). The zygotes were incubated and surviving two-cell-stage embryos were subsequently transferred into the oviducts of pseudo-pregnant female ICR mice.
4
Precise CRISPR-Mediated Mecp2 Editing
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Two CRISPR RNAs (crRNAs) and ssODNs with 5′- and 3′-homology arms (each 60 mer) flanking loxP variants (lox66 or lox71) were designed to target Mecp2 intron 2 and 3, based on our previous report (Table 1 ) [10 (link)]. To facilitate the detection of correct insertions, the ssODNs were engineered to contain an NheI restrictionsite and an EcoRI restriction site, respectively, in addition to the loxP sequences. Equal volumes of crRNA (100 μM; IDT, Coralville, IA) and trans-activating crRNA (tracrRNA) (100 μM; IDT) were combined in a duplex buffer (IDT), heated in a thermal cycler to 95 °C for 5 min, and then placed for 10 min at room temperature according to the manufacture’s protocol. Combined crRNA/tracrRNA (3 μM) was mixed with recombinant Cas9 protein (100 ng/μL; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA, USA) and ssODNs (400 ng/μL) in Opti-MEM I (Life Technologies, Carlsbad, CA, USA), and the mixture was placed for 10 min at room temperature before EP.
5
Generation of HMGB-1 Knockout Cell Line
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HMGB-1 KO was performed according to a previously published method that targeted HMGB-1 (Kang et al., 2021a (link)). To establish the HMGB-1 KO cell line, CRISPR RNA (crRNA) targeting HMGB-1 (catalog number A35509, chr13:30463559–30463537) and tracrRNA were purchased from Thermo Fisher Scientific and annealed according to the manufacturer’s instructions. Lipofectamine CRISPRMAX Cas9 transfection reagent (Thermo Fisher Scientific) was used to transfect HuARLT cells with CRISPR RNA and TrueCut Cas9 protein V2. After 3 days, the cells were detached with trypsin–EDTA and counted at the desired concentration. The cells were then seeded into a 96-well culture dish with 0.8 cells per well and 100 μL of medium for cloning. Single clones were analyzed by western blotting and sequencing to confirm HMGB-1 KO. The HMGB-1 KO clone was cultured in a large culture dish and used in subsequent experiments.
6
Isoform-specific CRISPR knockout of Trp73 in mice
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The CRISPR/Cas9 system was utilized for isoform-specific knockout of the Trp73 gene in C57BL/6J mice. Two types of small RNAs, target-recognizing CRISPR RNA (crRNA) designed with CHOPCHOP (48 (link)), and auxiliary transactivating crRNA (tracrRNA), were purchased from FASMAC as listed in Supplementary Table S3. We delivered TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) together with the crRNA and tracrRNA into in vitro–fertilized eggs of C57BL/6J mice according to the institutional procedure of Kyoto University (49 (link)). Both TAp73 and DNp73 knockout genotypes were obtained with 1bp insertions on the crRNA recognizing region, which results in truncating (frameshift) mutations (Supplementary Fig. S4A). Genotyping for each knockout isoform was performed with restriction enzyme digestion of PCR-amplified DNA. For TAp73, genomic DNA was amplified with TAp73-genotype-F and TAp73-genotype-R primers (Supplementary Table S3), followed by BstBI (New England Biolabs) digestion, whereas DNp73-genotype-F and DNp73-genotype-R primers (Supplementary Table S3), and MseI (New England Biolabs) were similarly utilized for DNp73 genotyping.
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