How can I design sgRNA (tracrRNA) for CRISPR / Cas9?

Jiho Seok @Jiho-Seok

14 February 2023 0 2K Report

Hi, I'm trying to use CRISPR/Cas9 system with E. coli.

I totally understood the way to design my crRNA sequence for my target.

However, one question is how can I design "tracrRNA sequence?"

I mean, when we design the sgRNA, crRNA can be changed but tracrRNA scaffold have same sequence.

How can I know this sequence?

Thank you !!

Badges
Science method

More Jiho Seok's questions See All

Do you know what kind of bacteria it is?

Medium Yeast Ext Glucose Na-acetate Tween80 Fe, Mg, Mn, phosphate Please let me know. Thank you in advance.

16 May 2023 8,043 1 View

How does one quench the Surfate based AOP reaction before determining the TOC?

My experiment is Surfate based AOP with Co, Cu, Fe catalyst. I know that Methanol is usally added to quench the reactoins, however, it has some problems when analyzing the TOC I tried to use KI...

28 November 2021 6,732 1 View

Transfecting Plasmid DNA into THP-1 Cells using FuGene HD vs. Lipofectamine LTX?

We are trying to transfect plasmid DNA into the THP-1 and primary monocytes. However, transfection efficiencies of lipofectamine 2000 and Genjet were not enough to perform subsequent downstream...

21 May 2021 1,628 1 View

Bacteria culture problem?

I transformed lentiviral DNA into DH5a and cells grow well on the agar plate. But they don't grow in LB media after inoculation. It worked well with Dh5a in my previous lab although the company...

18 April 2021 8,903 1 View

Paradox of explicit rating for collaborative filtering-based recommendation system?

In the process of investigating the effect of negative cases on model performance using the Movielens 100k dataset, I’ve got a question. I did two experiments to evaluate model performance. In the...

23 March 2021 7,010 0 View

Does DTT affect cell lysis?

Hi, I am currently working with S. cerevisiae. general rule of thumbs for yeast cell lysis is to use 0.2M NaOH incubation followed by lysis using high urea buffer (containing 100mM DTT) at 70oC...

05 September 2020 7,244 3 View

How can I disaggregating silica nanoparticles(~22nm) agglomerates?

We purchased some silica nanoparticles (~22nm, 34 wt%)dispersed in water from Sigma Aldrich , and planned to mix this nanoparticles with GPTMS( (3-Glycidyloxypropyl)trimethoxysilane) and distilled...

01 June 2020 781 5 View

How to make the construction of knock-in vector?

Dear Researchers! I want to make the knock-in vector to construct the knock-in cell line. But my ligation experiment dose not work (Always backbone vector was self-ligation) I just let you know...

23 November 2019 9,833 5 View

Experiment conditions for ELISA using the purified protein and synthetic peptide?

Hi. I'm planning to perform ELISA to find amino acid residues which directly bind to the protein X (protein of interest). You may see the design of the experiment in the picture that I posted....

07 August 2019 2,742 3 View

Does anyone use deuterated methanol in 13C-NMR analysis? Supposingly it should have 7 peaks around 49.3ppm, but why my results only got 2 peaks?

My sample is a pure 3-MCPD ester.

27 March 2018 7,687 9 View

MAGeCK Plotting Error: Logarithmic Axis Must Have Positive Limits?

I have been running a MAGeCK test command on the terminal for a CRISPR screen to rank sgRNAs and genes based on the read count tables. However, I get only the plot of the top-ranked genes...

28 July 2024 9,811 1 View

Why knockouts for same genes from two different gRNA are showing different result?

Why knockouts for same genes from two different gRNA are showing different result? I followed CRISPR-CAS9 gene editing technology.

25 July 2024 8,313 1 View

Is possible to edit a gene present in a plasmid by Crispr Cas9?

Hello everyone! Someone working with Crispr Cas9. I have a question. Is it possible to edit a gene that is present on a plasmid (in this case with a low copy number)? In this case, is it possible...

22 July 2024 7,073 2 View

How to activate 4E8 T cells with plate-bound anti-CD3 for a genome-wide CRISPR screen?

It was assumed that 4E8 T cells should be isolated and activated using plate-bound anti-human CD3, after which would be used for a genome-wide CRISPR screen. (Ref: Genome-wide CRISPR Screens in...

17 July 2024 3,371 0 View

What is the most effective method for knocking out a gene in Candida albicans?

I have attempted various methods, including homozygous gene disruption and CRISPR-Cas9, but have not achieved the desired results. It appears that the gene of interest is situated in a...

15 July 2024 3,977 1 View

What would you do next?

Studying a specific monogenetic disease based on mutations in different individual genes. I try to recreate the disease phenotypes of donors in vitro. I knocked out a gene x with above 90%...

12 July 2024 6,313 0 View

Can Invitrogen™ LIVE/DEAD™ Cell Imaging Kit (488/570) be used on fixed cells?

Can the kit from Invitrogen™ LIVE/DEAD™ Cell Imaging Kit (488/570) be used on fixed cells on a scaffold?

08 July 2024 7,914 3 View

Are there any information about protocols of CRISPR in leishmania strains?

I am looking for a CRISPR protocol for application in leishmania strains

08 July 2024 9,732 1 View

Is it possible to generate a (semi-)permanent "transcription bubble" within a replicable plasmid?

I was wondering if it is possible to form a permanent open "ssDNA bubble" similar to a transcription bubble (>13 nucleotides) within E. coli. These criteria are important: 1. Open ssDNA...

07 July 2024 8,275 5 View

"What is the best method and model for evaluating the mechanical and viscoelastic properties of a viscous material with multiple particle inclusions?

"What is the most accurate method and model for evaluating the effective mechanical and viscoelastic properties due to the inclusion of multiple types of particles in a viscous material, and which...

16 June 2024 9,139 1 View