CRISPR in RNA Wonderland

doudna_crispr_wide-cb9478286d39615ed64291ea95d4cfe022596aa2-s900-c85This week our journey among leading labs takes us to meet a pioneer of gene silencing. Pino Macino contributed to the birth of RNA interference, a field awarded a Nobel prize in 2006, and teaches cell biology at Sapienza University of Rome.  He thinks CRISPR is a great leap forward in understanding the function of genes.

macinoYou are one of the fathers of the old technique that is being overshadowed by the new one, therefore your opinion is twice as valuable. Are you converting to CRISPR?

We are using it to explore the relationship network of micro-RNAs, a large family of small molecules acting as regulators. This technique is a wonderful tool for basic research. It doesn’t mean that RNA interference is outdated, as it is still useful for certain studies, but CRISPR is going to answer plenty of new questions.

Can you compare the two techniques?

If you want to study a gene, the first step is to silence it, i.e. disabling the gene itself or the product of its expression, and then see what happens. Interference works by introducing a tiny RNA molecule (20-25 letters) complementary to the RNA messenger that is the transient product of gene expression. They bind and this double-stranded molecule is recognized by a cutting enzyme called Argonaute. The gene transcript, therefore, is destroyed and the gene is silenced.

The CRISPR platform employs a protein of the Cas family instead. What’s the difference?

It directly cuts DNA rather than RNA opening up new possibilities. For example, there are mice expressing Cas9 in every cell, or only in certain tissues, making pretty easy to introduce targeted mutations into the genome. You can fix a faulty gene or mutate a healthy one, and the effects are going to be passed down from one generation to the next. What you get is a stable line of mice to be used as a model of human disease. It will be done also on humans in the future, maybe not only for therapeutic purposes but also for enhancement. But this scenario is far enough out in time that I won’t be there and I’m relieved not to have to confront these ethical problems.

So the CRISPR game is more engaging and lasting, which application is particularly promising in your opinion?   

Lots of exciting things can be done by using Cas proteins adapted for targeting specific sequences without cutting. The tool stops working as molecular scissors and can be coupled to epigenetic modifiers in order to add or remove chemical marks from histones, the proteins that package DNA into chromosomes, influencing whether and how genes are expressed. Future drugs working this way will be amazing.

The invention of CRISPR was inspired by strange repeats found in bacterial genomes and the critical insight came from bioinformatics. How important will be computer science in the CRISPR era?

Last year the Sapienza University started a 3 years degree in bioinformatics for medicine, engineering, and natural sciences.  CRISPR is going to make this approach more and more useful. Think of libraries of edited human cells for example, with mutations covering the entire genome. This load of data will need bioinformatics to be interpreted.

DNA has obscured RNA, in research and storytelling, for a long time. Is it still the case?

RNA was considered as an object, to be used and then thrown away. Today it has become a subject. There are tens of thousands of RNAs that do not code for proteins but do the work of nucleic acids, taking three-dimensional shapes that can be readily tested by evolution. RNAs interact with DNA, between them and with proteins, altering their properties, but we still know too little about them. By mutating non-coding DNA regions, CRISPR will also help decipher this complex micro-world. Amazing discoveries are likely to come.


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