Tracrrna

Manufactured by Integrated DNA Technologies

Sourced in United States

TracrRNA is a component of the CRISPR/Cas9 gene editing system. It functions as a guide RNA that forms a complex with the Cas9 enzyme, directing it to the target DNA sequence for cleavage.

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Lab products found in correlation

68 protocols using tracrrna

CRISPR-mediated Hand2 gene editing in mice

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The guide RNA sequences (5′-CGCTGAGAGCCCTTTGCACA-3′ and 5′-AATAATTATTGAGCGCAGCT-3′) were designed to target DNA sequences upstream of the Hand2 start codon. The 2 crRNA solutions were mixed with equal volumes of 200 mM tra crRNA (Integrated DNA Technologies) separately, annealed into crRNA-tra crRNA duplexes (95°C, 5 minutes), and cooled to room temperature; 2 ml of each crRNA-tra crRNA duplex solution was mixed with 1.1 ml Streptococcus pyrogenes (S.p.) HiFi Cas9 nuclease (Integrated DNA Technologies) and 48 ml of advanced KSOM Medium to assemble the ribonucleoprotein complex (65 (link)). Hormonally stimulated C57LB/6 female mice were mated to C57LB/6 males, and zygotes were collected from oviducts at E0.5 and washed and transferred into advanced KSOM Medium. The ribonucleoprotein complex solutions were mixed and injected into the zygotes in advanced KSOM Medium. Injected zygotes were cultured in KSOM (37°C, 5% CO₂) to the blastocyst stage. The blastocyst embryos were transferred into the uteri of pseudopregnant ICR female mice on D3.

Xin Q., Feng I., Yu G, & Dean J. (2024). Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in the mouse uterus. The Journal of Clinical Investigation, 134(5), e174194.

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CRISPR-Mediated Reg Knockout Mouse

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CRISPR/Cas-mediated genome engineering was conducted to create Reg deficient mice. To target intron1 of the mouse Reg3b and Reg3g genes, gRNA for both murine Reg3b and Reg3g were designed using CRISPR direct (https://crispr.dbcls.jp). The sequences of the guide for target DNA were as follows: Reg3b -G (5′-CTG TCT TTC TCC TGT GAT AC-3’) and Reg3g -G (5′-AAG AAG GGG AGA ATT AGT GT-3′), which were located at intron1 of both Reg3b and Reg3g, respectively. The corresponding crRNA, tracrRNA, and Cas9 nuclease were purchased from Integrated DNA Technologies (IDT). The crRNA and the tracrRNA were annealed and complexed with Cas9 nuclease according to the manufacturer’s instructions.

Chen W., Imasaka M., Lee M., Fukui H., Nishiura H, & Ohmuraya M. (2023). Reg family proteins contribute to inflammation and pancreatic stellate cells activation in chronic pancreatitis. Scientific Reports, 13, 12201.

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Preparation of Dual-guide RNAs for CRISPR

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Dual-guide RNAs were prepared as described previously (13 (link)). Briefly, DNA oligonucleotide templates for crRNA and trans-activating CRISPR RNA (tra crRNA) were purchased from Integrated DNA Technologies (IDT). The crRNA template sequences were as follows: 5′-TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTG-3′, where the 20 Ns represent the 20 nucleotide target regions (Table S2). The tra crRNA template sequence was as follows: 5′-TAATACGACTCACTATAGGACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT-3′. Then, crRNA templates for every target were pooled. The tra crRNA and pooled crRNA templates were then transcribed using T7 RNA polymerase (NEB) for 2 h at 37°C. crRNAs and tra crRNAs were purified using AMPure magnetic beads and then annealed together at an equimolar ratio to form 40 μM dual-guide RNAs. These dual-guide RNAs were stored at single-use aliquots at −80°C for a year. Immediately prior to use, the dual-guide RNAs were annealed at 95°C for 30 s, then allowed to cool at room temperature.

Tram T.T., Ha V.T., Trieu L.P., Ashton P.M., Crawford E.D., Thu D.D., Quang N.L., Thwaites G.E., Walker T.M., Anscombe C, & Thuong N.T. (2023). FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum. Journal of Clinical Microbiology, 61(4), e01634-22.

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CRISPR-Cas9 Mutagenesis in P. pacificus

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We used the modified CRISPR-Cas9 protocol for P. pacificusas previously described (Witte et al., 2015) with hybridized target-specific CRISPR RNAs (crRNAs) and universal transactivating CRISPR RNA (tracrRNA) obtained from Integrated DNA Technologies (Alt-R product line) for generating mutants. The sgRNA and all the crRNAs targeted 20 bp upstream of protospacer adjacent motifs (PAMs).
To hybridize crRNA and tracrRNA, 10 µl of the 100 µM stock of each molecule were combined, followed by a denaturation step at 95°C for 5 minutes and annealing at room temperature for another 5 minutes. Five µl of the hybridization product was combined with 1 µl of 20 µM Cas9 protein (New England Biolabs) at room temperaturefor 5 minutes. The mixture was diluted with Tris-EDTA buffer to a total volume of 25 µl and injected into the gonad of young adults. Molecular lesions were detected in F1 progeny by Sanger sequencing.

Lenuzzi M., Witte H., Riebesell M., Rödelsperger C., Hong R.L., & Sommer R.J. (2021). Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

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CRISPR-Cas9 Targeting of Anolis sagrei Tyrosinase Gene

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The CRISPOR target selection tool (version 4.4) was used to select target regions with efficiency scores of 50% or greater within the second exon of the A. sagrei tyr gene (Haeussler et al., 2016 (link)). The tyr gene reference sequence was obtained from a draft genome assembly of Anolis sagrei. Alt-R CRISPR-Cas9 crRNAs, tracrRNA, and Cas9 V3 were purchased from Integrated DNA Technologies, Inc. The crRNA target sequences were as follows:

AsagTyrEx2A: 5′-TTGCCGGGGTTTCGAAGAAT-3′

AsagTyrEx2B: 5′-ATGATAAAGGGAGGACACCT-3′

AsagTyrEx2C: 5′-GAAGTTAGCCATTTTGTCCA-3′

Cas9 RNP was prepared by following manufacturer recommendations. A 5 μM injection solution was made using a standard microinjection solution (10 mM Tris-HCl, pH 7.4) containing phenol red to help verify that injected solutions entered the oocytes.

Rasys A.M., Park S., Ball R.E., Alcala A.J., Lauderdale J.D, & Menke D.B. (2019). CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes. Cell reports, 28(9), 2288-2292.e3.

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CRISPR-Cas9 Ribonucleoprotein Complex Formation

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CRISPR RNA (crRNA), transactivating RNA (tracrRNA), and Cas9 from S. pyogenes were purchased from Integrated DNA Technologies (IDT). crRNA sequences were selected from the IDT or GenScript® database (SI Appendix, Table S1). Single crRNA and tracrRNA were dissolved in nuclease-free duplex buffer (IDT) and mixed in a 1:1 molar ratio (210 pmol). The mix was heated to 95 °C and cooled down Δ0.1 °C/s until RT. The resulting gRNAs were either mixed individually or pooled together with Cas9 at a 3:1 molar gRNA:Cas9 ratio and incubated for 30 to 60 min at RT. Electroporation enhancer (IDT) was added to the ribonucleoprotein (RNP) complexes and nuclease-free duplex buffer, if required, to achieve a final volume of 10 µL per reaction. As negative control, RNPs containing crRNA#1 (IDT) were prepared as described above. If not used immediately, RNPs were stored at −20 °C.

Barends M., Koller N., Schölz C., Durán V., Bošnjak B., Becker J., Döring M., Blees H., Förster R., Kalinke U, & Tampé R. (2023). Dynamic interactome of the MHC I peptide loading complex in human dendritic cells. Proceedings of the National Academy of Sciences of the United States of America, 120(25), e2219790120.

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MANF Gene Deletion in Stem Cells

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To recapitulate the gene mutations found in the patients, guide RNAs targeting the first exon of MANF gene for deletion were designed using Benchling (https://benchling.com). Guide RNAs with the highest quality score and lowest off-target score were selected and purchased, alongside the RNP components (HiFi Cas9 protein, crRNA, and tracrRNA), from Integrated DNA Technologies and used according to the manufacturer’s recommended protocol. Two million undifferentiated stem cells were electroporated with the RNP complex using the Neon Transfection system (1,100 V, 20 ms, 2 pulses) (Thermo Fisher Scientific) and plated on Matrigel-coated plates in Essential 8 medium containing 10 μmol/L ROCK inhibitor overnight. Afterward, cells were single-cell sorted, expanded, and screened for the desired deletion using PCR. Positive clones were validated by Sanger sequencing and characterized for pluripotency and chromosomal integrity.

Montaser H., Patel K.A., Balboa D., Ibrahim H., Lithovius V., Näätänen A., Chandra V., Demir K., Acar S., Ben-Omran T., Colclough K., Locke J.M., Wakeling M., Lindahl M., Hattersley A.T., Saarimäki-Vire J, & Otonkoski T. (2021). Loss of MANF Causes Childhood-Onset Syndromic Diabetes Due to Increased Endoplasmic Reticulum Stress. Diabetes, 70(4), 1006-1018.

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Generation of MANF Gene Knockout Stem Cells

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To recapitulate the gene mutations found in the patients, guide RNAs targeting the first exon of MANF gene for deletion were designed using Benchling (https://benchling.com). gRNAs with the highest quality score and lowest off-targets score were selected and purchased, alongside the RNP components (HiFi Cas9 protein, crRNA and tracrRNA), from Integrated DNA Technologies (IDT) and utilized according to the manufacturer’s recommended protocol. Two million undifferentiated stem cells were electroporated with the RNP complex using Neon Transfection system (Thermo Fisher, 1100 V, 20ms, two pulses), and plated on Matrigel-coated plates in E8 medium containing 10 μM ROCK inhibitor overnight. Afterwards, cells were single-cell sorted, expanded, and screened for the desired deletion using PCR. Positive clones were validated by Sanger sequencing and characterized for pluripotency and chromosomal integrity.

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Genetic Engineering of dCas9-HDAC1 Fusion Protein

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The full-length HDAC1 cDNA was obtained by PCR using a plasmid expressing Flag-tagged HDAC1 (gift from Dr. Ed Seto’s lab at H. Lee Moffitt Cancer Center, Tampa, FL) as template and constructed into pcDNA3.1-dCas9 (Addgene plasmid #47106, a gift from Charles Gersbach) (27 (link)) expression vector to express dCas9-HDAC1 as a Flag-tagged protein. A His to Ala mutation (CAT to GCT) at position 141 of the HDAC1 protein sequence was introduced into pdCas-HDAC1 plasmid using Q5® Site-Directed Mutagenesis Kit (New England BioLabs, E0554) to generate pdCas9-HDAC1 enzymatically dead mutant (pdCas9-HDAC1mt). The exponential amplification by PCR was using primers (5’-GGGCCTGCACGCTGCAAAGAAGTCC-3’ and 5’-CCAGCCCAATTCACAGCG-3’). The pdCas9-HDAC1mt was confirmed by nucleotide sequencing (MCLAB, CA, USA). pBabe-Kras G12C was a gift from Channing Der (Addgene plasmid #58901). CrRNAs targeting human KRAS promoter were designed using Broad Institute online tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). The following crRNAs sequences were used: crRNA 1, 5’-UCUUCAGACGGGCGUACGAGGUUUUAGAGCUAUGCU-3’, crRNA 2, 5’-CAGGGACUUCGCUUAUACCCGUUUUAGAGCUAUGCU-3’, crRNA 3, 5’-AUCAUCACGACAACCUUAUGGUUUUAGAGCUAUGCU-3’. CrRNAs and tracrRNA were purchased from Integrated DNA Technologies (IA, USA).

Liu J., Sun M., Cho K.B., Gao X, & Guo B. (2020). A CRISPR-Cas9 repressor for epigenetic silencing of KRAS. Pharmacological research, 164, 105304.

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Efficient CRISPR/Cas9 and Base Editing in HEK293 and hiPSCs

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HEK293 cells were transfected in 24-well multi-wells with 500–750 ng of Cas- or ABE-encoding plasmids, 250 ng of the desired pUC19-sgRNA plasmid and, in HDR experiments, 200–500 ng of ssODN using TransIT-LT1 (Mirus Bio), according to manufacturer’s instructions.
A total of 2 X 10⁵ HEK293/CdLS-clones (program CM-130, solution SE) and patient-derived hiPSCs (program CM-113, solution P3) were electroporated on a Lonza Nucleofector 4-D according to manufacturer’s instructions. Briefly, equal amount of 100-µM crRNA and tracrRNA (ordered from Integrated DNA Technologies) were mixed together to form gRNAs. 150 pmol of gRNAs were complexed with 120 pmol of Cas9 proteins (from Integrated DNA Technologies) to form RNPs. Electroporation mix was prepared as previously described [26 ]. When used, 1 µM of NU7441 (Selleck Chemicals, Cat# S2638) was added to the fresh medium on day 1 and day 2 after the electroporation.

Umbach A., Maule G., Kheir E., Cutarelli A., Foglia M., Guarrera L., Fava L.L., Conti L., Garattini E., Terao M, & Cereseto A. (2022). Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology. Stem Cell Research & Therapy, 13, 440.

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