Targeting Huntington at CattaneoLab

huntingtonCRISPR is radically changing the way researchers work, by allowing better, faster, and cheaper experiments. This blog will tell, among other things, how leading labs are using the most popular technique for genome editing. Let the dance begin with the Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases of the University of Milan (CattaneoLab). The group directed by Elena Cattaneo is busy unveiling the molecular basis of neurodegeneration in Huntington’s disease with the help of CRISPR, as pharmacologist Chiara Zuccato explains.

zuccato“We began using CRISPR in 2015, it’s prodigious, phenomenal and it has transformed our experimental strategy. Huntington is a brain disorder caused by mutations in the gene coding for huntingtin, a protein important for neurons development and function. Expansion of the trinucleotide CAG repeat in the mutated gene results in a toxic version of the protein harming neurons and causing the disease. We are interested in studying the gene’s functions and verifying if they are impaired by mutations. This is why we introduce targeted mutations into the gene in mice and also human stem cells. Then we check if stem cells are able to correctly differentiate into neurons. To sum up we investigate correlations between structure and function.

What upsides do you get out of CRISPR?

“When I was taking the first steps in the lab in 1999, two to three years were needed to engineer the desired gene and success was not guaranteed. It was done by homologous recombination, the technique for which Mario Capecchi was awarded a Nobel prize. That approach however is cumbersome and quite inefficient. With CRISPR we can do it in barely a month. A couple of weeks to receive the customized kit including the cutting enzyme (Cas9) and its RNA guide, plus 2 or 3 weeks for the experiment. Three colleagues (two senior researchers and a graduate student) are assigned full time to the task of rewriting portions of the Huntington gene by means of CRISPR.

How did you get by before CRISPR?

“Homologous recombination is hard to use and not always successful, therefore we used to employ small circular DNA molecules (plasmids). They integrate into random sites in the genome, carrying the gene with the desired mutations (transgene). As a result the cell keeps its endogenous gene and also acquires the foreign one. It’s a valuable approach to study the function of genes but not really representative of physiological conditions, because the transgene random insertion may disrupt the sequence of other genes. We needed several clones of the engineered cells to be certain that our results were real, and we have always validated our data by involving other teams skilled in homologous recombination. Nowadays we can do everything in a fraction of time by editing the endogenous gene with CRISPR without introducing alien DNA. Custom reagents for any particular experiment can be ordered online for as little as few hundred dollars. Useful tricks to improve experimental conditions are discussed in specialized blogs. There is an open community sharing information on the technique, making problems easier and speeding up discoveries.”

How do you choose which mutations to introduce?

“It’s based on an evolutionary study started about 5 years ago. There are no other genes similar to the Huntington’s gene in the human genome, therefore comparisons must be done with other species. We have a database of DNA from over 200 different organisms, from amoebas to monkeys. Huntingtin is a very big gene, encoding for a protein that is larger than 3,000 amino acids in size. By comparing different species we discovered that the first 500 amino acids are the most interesting part and are sufficient to carry out functions important for neurons. We target this region in order to understand if it includes smaller portions, even single amino acids, that are essential for these tasks.

How does the CRISPR complex enter the cell?         

“We put the enzyme Cas9 and its RNA guide into vesicles called liposomes, the mixtures is then spread over the cells. Liposomes fuse with the cell membrane and their content is released. Cas9 and the guide eventually reach the DNA site to edit.”

What about CRISPR’s limitations?

“Sometimes the DNA is edited also at undesired locations, that may have similar sequences, but it rarely happens because the technique is really specific. When we think it may have happened we sequence the genome’s cell after editing and check for off-target effects. Now it’s affordable.

The technique is continuously being refined to perform new tasks with growing precision, have you tried only the classic Cas9 enzyme?

“ We have also used other variants, including the one that finds the target sequence without cutting the DNA. It’s called dead-Cas9 and can be equipped with proteins turning genes on or off. This is particularly useful when the aim is to modulate the expression of a gene instead than introducing mutations. Different CRISPR variants are suitable for different situations and we can choose from multiple options, which is great.

(photo: neurons transfected with a disease-associated version of huntingtin)

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