Product Survey: Genome Editing Kits

30 Years in the Making
by Harald Zähringer, Labtimes 02/2016

CRISPR-Cas9 and other targeted-nuclease related techniques took the sweat out of gene editing and made it available to ordinary biologists. But they are still based on concepts developed in the nineteen-eighties, by the pioneers of gene targeting in embryonic stem cells.

“The potential now exists for modifying any gene, in a defined manner, in any species from which functional ES cells can be obtained”.

Nope, that’s not a quote taken from one of the countless CRISPR-Cas9 papers currently flooding the scientific literature. It’s a far-sighted statement made by Nobel Prize winner and genetic engineering pioneer, Mario Capecchi, in a 1989 Science paper, referring to the then newly-developed, gene targeting technology in mice, based on homologous recombination. Albeit the current ballyhoo about CRISPR-Cas9 and other nuclease-related, genome editing techniques may leave another impression: gene targeting or editing is not a new concept. It already started in the early days of modern molecular biology, back in the nineteen-eighties – long before commercial gene editing kits were available.

Disrupted genes

In 1987, Mario Capecchi’s group at the University of Utah, developed a method to specifically knock out targeted genes in embryonic stem (ES) cells. Inspired by yeast cells, which easily integrate exogenous vector-DNA into homologous sites, Capecchi’s group microinjected embryonic mouse stem cells with a copy of the hypoxanthine phosphoribosyl transferase gene (hprt) that contained a clever modification: it harboured the gene encoding neomycin phosphotransferase (neo), which disrupted the coding sequence of hprt and rendered cells that incorporated the construct via homologous recombination ­resistant to the drug G418. Capecchi collected the few cells that grew on G418 medium, injected them into a mouse blastocyst and implanted the blastocyst into the uterus of a foster mouse.Since the resulting offspring were chimeras with cells derived from the host blastocyst and the modified ES cells, several rounds of breeding were necessary to get homozygous mice harbouring the ­mutated gene.

Gene targeting pioneer Oliver Smithies showing a sketch of his concept to knock out genes in embryonic stem cells by homologous recombination. Photo: Volker Steger

At about the same time, Oliver Smithies, who shared the Noble Prize in Physiology or Medicine with Capecchi and Martin Evans in 2007, applied a similar technique in his lab at the University of Wisconsin-Madison. In contrast to Capecchi, however, who had disrupted hprt, Smithies team corrected a defective hprt Gene in ES cells with an insertion vector harbouring the functional allele.

Clean deletions

The targeted gene disruption technique of Capecchi and Smithies was further refined by other groups. But a major issue of this approach still persisted: the neo cassette remained embedded into the host’s genome, which in turn gave rise to unwanted and unpredictable side effects on gene expression or other cellular functions.

This problem was solved a few years later by Klaus Rajewsky’s group, then at the University of Cologne. Rajewsky needed “clean” gene deletions, cleared from the neo cassette to study the immune response in mice. Hence, he came up with the idea, to combine Capecchi’s gene targeting strategy with the Cre-loxP recombination system, derived from the bacteriophage P1.

The Cre-loxP system is based on P1’s site-specific recombinase Cre that cuts target DNA at specific loxP recognition sites. To get rid of the neo gene, Rajewsky incorporated three loxP sites into the gene-targeting vector at strategical positions: two are placed at the flanks of the neo cassette and the third is installed further upstream.

Floxed mice

This construct is transferred into ES cells and integrates into the target gene via homologous recombination. In the next step, Cre is transiently expressed to excise the neo cassette. Since Cre leaves one loxP site of the former neo cassette behind, the neo-cleared target gene is now flanked by two loxP sites. The modified ES cells are then implanted into foster mice to create “floxed” mice. Crossing homozygous, floxed animals with mice expressing Cre, finally leads to Cre-loxP knock-out mice.

The Cre-loxP strategy is still very popular to alter and delete genes in mice and has also been implemented into other model organisms, such as yeast, plants or mammalian cell cultures. It is, however, a very labour-intensive technique, which requires different animal lines (Cre and loxP). Besides that, it lacks the possibility to introduce point mutations into the targeted genes.

The age of targeted nucleases

No wonder, then, that researchers enthusiastically hurrahed easier, faster and more versatile gene editing approaches, based on targeted nucleases, namely Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeat-associated Protein 9-nucleases (CRISPR-Cas9), which entered the scene only recently.

The basic concept and mode of action of targeted nucleases is very straightforward and pretty much the same for ZFNs, TALENs and CRISPR-Cas9: the nuclease is guided by a specialised protein (ZFN and TALENs) or RNA-molecule (CRISPR-Cas9) to a specific site of the target sequence and introduces a double-strand break (DSB) into the DNA.

The cellular DNA repair machinery has two options to fix the DSB: either by error-prone non-homologous end joining (NHEJ) in the absence of a homologous sequence, or through accurate homology-directed repair (HDR) in the presence of a homologous template.

Fast and easy gene editing

The NHEJ process usually introduces InDels (insertion/deletions) into the fixed DNA, which commonly provoke a frameshift downstream of the DSB that finally knocks out the target gene. The NHEJ mechanism is a nice-to-have feature that cells offer to molecular biologists. But the real gift of Mother Nature is the homology-directed repair pathway that allows fast and easy gene editing. To introduce, for example, a point mutation into the target gene, an exogenous DNA template with homologous arms flanking both sides of the modified sequence is transferred into the cell, together with the favoured nuclease system that executes the necessary DSB breaks.

CRISPR takes the lead

The forerunners of targeted nucleases, ZFN and TALENs, are based on protein-guided Fok1 nucleases. They are still widely used and favoured by some researchers in certain applications, however, the design of the guide proteins is still pretty complex and time consuming.

Hence, many researchers immediately jumped on the CRISPR-Cas9 bandwagon that departed in 2012, promising easy gene editing to the ordinary biologist in no time. And things are getting even easier with commercially available CRISPR-Cas9 gene editing kits.

They usually contain a suitable vector, expressing the necessary CRISPR-Cas9 components, which are exactly two: Cas9 and the single guide (sg) RNA that directs Cas9 to its target. The only thing customers have to do is to clone target-specific ­oligos in between the promoter that drives the sgRNA expression and the sgRNA scaffold.

What took Capecchi and Smithies years of hard work and caused them quite a few headaches, may be accomplished with a CRISPR-Cas9 kit in a few days or weeks, without having to think too hard.

Still working at the bench

By the way, both Capecchi and Smithies are alive and kicking and still run their own labs – Capecchi at the age of 79, Smithies at the age of 90.

Though already in his nineties, Smithies is well aware of CRISPR-Cas9 and the recent developments of targeted nucleases. In an interview conducted by Jane Gitschier from PLOS in 2015, Smithies commented on CRISPR, “I’m delighted to see [that things are so quick], because the old method was very laborious. I think CRISPR opens the possibility that people will be able to use homologous recombination in many ways. They’ll probably be able to use it to correct genes, and that was what I hoped to be able to do 30 years ago. It’s delightful.”

First published in Labtimes 02/2016. We give no guarantee and assume no liability for article and PDF-download.

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