Blog

María Rodríguez-López and Cristina Cotobal, Postdoctoral Fellows in Jürg Bähler’s lab at University College London recently published a very detailed Method Article on a new CRISPR/Cas9-based protocol and primer design tool. We asked them to explain what CRISPR/Cas9 is, what it can be used for and why they decided to publish their article on our platform.

 

What is CRISPR/Cas9 and what can it be used for?

In a nutshell, CRISPR/Cas9 is a complex that can identify and then cut pieces of DNA. It is derived from the bacterial “immune system” and can be used as a genome editing tool, by directing the endonuclease protein Cas9 by an RNA guide to specific genomic sequences to introduce DNA breaks. You could picture this technique as microscopic scissors that find specific “words” in our genetic “book” and are able to remove or replace these “words” with new ones, like a find-and-replace for the genetic instruction manual. Cells can then repair the DNA breaks either by introducing mutations or, if a template is provided, by replacing the target DNA with the template.

CRISPR/Cas9 is a game-changer for biological research, because it allows for precise, marker-free modification of DNA in any organism, with the potential to be used in human gene therapy in the near future. The technology is constantly evolving, with the discovery of new enzymes and development of technical variations that make this approach more specific with fewer side effects.

 

 

Why did you decide to write such a detailed protocol on the method?

We believe our detailed protocol will be very helpful to the fission yeast community for ‘seamless’ genome editing. Our protocol will also help to reduce the experimental time required for the whole process.

Traditional research or methods papers often do not allow for the provision of such details. But the devil is in the detail which make the difference to success.

 

Is it the first time CRISPR has been used in fission yeast; why is this significant?

This is not the first time that CRISPR has been used in fission yeast, but we have optimised a previously published protocol and introduced several changes to the technique that make it more efficient. The previous protocol simply did not work well for us and many other colleagues, which was frustrating. In addition to the wet-laboratory protocol, we have developed a web tool that will reduce the time on the critical primer design process that sometimes can be daunting.

 

All data and software used in this study is available online. Why is Open Source Software and Open Data important?

For software, as for science, there is always scope for improvement, and making our software and data available allows for other users to improve it and add new modules. We believe in the collective advancement of science.

 

Why did you decide to publish this article on Wellcome Open Research?

Our current approach for CRISPR deletions in S. pombe is effective, and we have applied it to modify more than 80 regions on the fission yeast genome. However, there is always room for improvement, for example by introducing new variations to make cloning more rapid or streamlined.

In the fission yeast community, there has been an open debate about the need to make the CRISPR/Cas9 method more efficient. Wellcome Open Research allows updated versions of published papers to be added, which is ideal for a rapidly evolving protocol such as ours and will provide us with the flexibility to add fresh aspects of the protocol as and when available. We therefore thought that this is an ideal platform to publish it on.

With the rapid publication at Wellcome Open Research, we are providing the community without delay a method that can be applied straight away, yet can be updated with future improvements.

We think Wellcome Open Research offers an open, dynamic and interactive platform for researchers. This publishing model represents a fresh and innovative opportunity for early career researchers to rapidly communicate their research and establish new relationships with other researchers interested in the same field.

 

Would you encourage other Wellcome grantees to publish on Wellcome Open Research?

We highly encourage other Wellcome grantees to publish on this platform. Our experience was very positive, with short turnaround time and almost immediate publication. The review process is open and you can introduce changes to your article in the future if you feel that an addition would make significant improvements to it.

 

Dr. Cristina Cotobal received her PhD from the University of Salamanca, Spain. She worked as a postdoctoral fellow at the University of Cambridge, UK in Juan Mata’s laboratory, before joining HelixNano, a biotech star-up in collaboration with Johnson-Johnson pharmaceutical, where she worked on the development of new techniques to improve the CRISPR/Cas9 approach in mammalian cells lines. She is currently appointed as a postdoctoral associate in Jürg Bähler’s lab at UCL and is interested in the regulation of long non-coding RNA expression under distinct cellular differentiation using CRISPR/Cas9 in S. pombe. 

  

Dr. María Rodríguez-López received her PhD in Genetics and Cellular Biology from the University Complutense of Madrid in 2013. For the past 3 years she has been working as a postdoctoral associate in Jürg Bähler’s lab at UCL. She is interested in the regulation of gene transcription, and applies a wide range of techniques to study this process in fission yeast. Currently she is using CRISPR/Cas9 technology to investigate the function of long non coding RNAs in this model organism.