Simple and effective CRISPR CAS9 gene editing for primary cells
Vector-free CRISPR-CAS9 gene editing to accelerate therapeutic applications
A few years ago, Ayal Hendel et al (doi:10.1038/nbt.3290) published results revealing that chemical alterations to sgRNA enhance gene editing in primary cells. To demonstrate this, Matthew H Porteus’s team chose the targeted genes CCR5, HBB and IL2RG respectively involved in anti-HIV clinical trials, cell anaemia and thalassemia, and severe combined immunodeficiency. More recently, the same team tested several modified CAS9 mRNA. You can find the practical results on this poster introduced at the ASGC.
Chemically modified guide RNA with CAS9 mRNA in human primary cells
2′-O-Methyl 3′ Phosphorothioate and 2′-O-Methyl 3’thioPACE incorporated at 3 terminal nucleotides at both 5′ and 3′ ends of the sgRNA increase the indel frequency so essential to knock-out. They even increase the level of homology-directed repair required for knock-in. These chemically modified sgRNA are recommended especially on primary cells.
They were shown to bring the following benefits:
- Increased gene editing efficiency (2x to 6x times more)
- No toxicity thanks to high pure preparation (HPLC+PAGE)
- Enabling of highly active RNA-only CRISPR-CAS9 delivery, avoiding the drawbacks of plasmids
U-depleted and 5MoU CAS9 mRNA is the top choice
This year, CAS9 has been the focus of Matthew H Porteus’s team. Several modifications were tried and compared as show on the figure above. Their work reveals that the top choice is a CAS9 mRNA that is U-depleted and modified with 5-methoxyuridine (5MoU). It is even far better than using CAS9 protein, through CAS9 RNP complex.
Keep in mind that useful CAS9 mRNA is available as catalogue product, thanks to Trilink’s expertise. Furthermore, it benefits from the CleanCap technology, offering more than 90% of capping. The capping is also a CAP 1 structure that boosts translation in Mammalian cells.
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Contact me for any questions, I’ll be pleased to offer advice on how to improve your CRISPR Cas9 gene editing with our broad range of tools.