Tip 1: Use an easy and cheap cell culture system to optimize your CRISPR experiments.

To screen for successful knockins, I used a quick and easy restriction fragment length polymorphism (RFLP) assay. I cloned each individual pair into the Cas9 nickase PX462 plasmid (Addgene #48131 for V1.0 and #62988 for V2.0) and co-nucleofected 1.5 ug of each construct together with 200 pmol 150-bp-long homology-directed repair (HDR) template into 1,000,000 HEK293s cells (more on HDR design here). Although wildtype Cas9 off-target effects in human pluripotent stem cells appear to be very low, I decided to be on the safest side possible and started off by designing single guide RNAs (sgRNA) for the less promiscuous Cas9 nickase system, focusing on only one of the three planned point mutations. It is time for another blog post of this kind, now with me in the writer’s seat, taking you through my CRISPR journey.Īnyone hoping to use CRISPR to establish a reliable cellular model of a specific human disease should have as their top priority the mitigation of off-target effects. However, this progress would have been impossible had it not been for the multitude of protocols that have been published across scientific journals and blogs around the web. Following multiple rounds of optimisations, trials-and-errors and long working hours, I have become a confident user of this technology. It is now April 2016, and I have come a long way. I had never used CRISPR/Cas9 and did not have a clue what conditions would work best in my hands!

I would generate these point mutations using CRISPR/Cas9, a technology providing scientists with state-of-the-art molecular DNA scissors. My project involved modelling disease-specific point mutations in human pluripotent stem cells. When I started my PhD project in October 2015, I knew three things for sure: Want to be Benchling’s guest blogger? Contact us. Check out this post for her tips and tricks! Ultimately, she was able to create point mutation knockins in HEK293s using CRISPR with up to 18% efficiency without selection. Below she shares her journey in harnessing the power of CRISPR. Editor’s note: This post was contributed by Ralitsa Madsen, a PhD student at the University of Cambridge.

Mutation Surveyor Demo