CRISPR genome editing: which cell line to choose?

Many labs have adopted the CRISPR genome editing technology to make knock-out and knock-in cell lines.

This technology produces first a targeted break in genomic DNA, which can then be exploited to produce cell lines with genes knocked out or where a donor vector has been used to introduce new genetic elements (point mutants, fluorescent tags, antibiotic resistance cassettes, etc.). Essentially any desired modification to the cells genome can be made. In setting up these genome editing projects there are many choices to be made including vector for the Cas9 protein and for the sgRNAs. Perhaps the most difficult choice, however, can be which cell line to use. Even the most affordable stable genome editing cell line development services can come with a significant cost, so choosing the right cell line at the beginning is crucial. Here we explain some of the choices researchers have in setting up their CRISPR genome editing projects and give our advice for cell line selection.

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Using CRISPR to knockout an essential gene

Genome editing technology enabled by CRISPR and TALEN has become mainstream. Most cell biology labs are engaged in projects to create custom cell lines with knock-outs and knock-ins, and companies such as GeneCopoeia even propose complete cell line generation services. Projects can involve transfection of mammalian expression constructs, TALEN pairs, or direct transfection of RNA.

When scientists want to make a stable knockout cell line, one of the first questions they should ask is whether or not the resulting cell line will be viable. Often a murine knockout mouse has been made and/or siRNA knockdown experiments have been performed in human cells, so experienced users have a good idea if a human knockout cell line will be viable. There are certainly some cases, however, where either the researcher knows or expects that a complete knockout will not be viable but wishes to make the knockout nonetheless. What is the best way to deal with all of the risk involved with starting an relatively expensive and time-consuming project like this that could end in failure?

To knockout an essential (or potentially-essential) gene we recommend the following:

1) Plan the entire project in collaboration with a professional company offering stable cell line development services.

2) Develop your knockout strategy using TALEN or CRISPR.

3) Introduce a functional copy of your gene under the control of doxycycline into a safe harbor knock-in site. This copy of the gene should be designed to be resistant to the TALEN/CRISPR knockout strategy designed in #2.

4) Generate the parental doxycycline-inducible/repressible cell line and isolate a stable clone.

5) Knockout the functional endogenous alleles of the gene in this stable clone.

You will be then left with a viable cell line that will allow you to test whether or not complete knockout results in viable cells. It may be even possible to include loxP sites in the knocked-in cassette to completely remove the doxycycline controlled wild-type gene.

safe_-harbor

 

Any questions? Leave your comments below…

Genome Editing in Stem Cells: outsource or do-it-yourself?

Many researchers are facing a dilemma: they want to set up a CRISPR genome editing project but they can’t decide which cell line to use for genome editing. Even some of the most cost-effective genome editing cell line generation services like the one from GeneCopoeia will cost a few thousand euros, so picking the correct cell line and setting up the project correctly is very important. Researchers working in primary cells may find the idea of switching to an immortalized cell line a bit artificial. They dream of the possibility of editing the genome of a stem cell line which they can then differentiate into their tissue of choice as needed. [Read more…]

Genome-Editing by Mail

Due to the development of CRISPR and TALEN technologies, the field of genome editing is evolving rapidly.  This post highlights the natural evolution of these technologies leading to outsourcing gene editing to skilled service providers.

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What’s Trending in the Molecular Biology World? CRISPR !!!

It’s official, CRISPR has officially reached “Breakout” status as a Google search term (see image from Google Trends). The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and RNA-guided Cas9 nucleases are an exciting new gene editing tool that allows life science researchers to directly modify the DNA of the organism they study.

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