Using CRISPR to elucidate the role of RIPK1 in tissue homeostasis
March 2015
The CRISPR/Cas9 system is revolutionizing the field of genome editing. Adapted from the bacterial immune system, CRISPR enables one to target and precisely alter any piece of genomic DNA. Used for years in the dairy industry to confer phage resistance in starter cultures, its utility was recently harnessed for use in eukaryotic systems. While mainstream media has touted this as the next big therapeutic approach, researchers have also been quick to adapt it to create unique transgenic animals rapidly and precisely.
Due to a variety of technical constraints, mouse models have long been the favored transgenic model. The traditional method of creating these mouse models through embryonic stem cell implantation is time intensive, requires multiple rounds of backcrossing, and still germline transmission is not guaranteed. Alternatively, with the CRISPR/Cas9 method embryos are modified directly, thus ensuring that the genetic modification is present in the germline. This eliminates months of backcrossing, saving time and money. This method also makes it more feasible to create other transgenic animals such as rats, mosquitoes, silkworms, cows, rabbits, zebrafish and monkeys. These benefits greatly improve and accelerate the progress of primary research.
In a recent article in Nature, Dannappel et al. take advantage of the CRISPR method to generate a new transgenic mouse model to help elucidate the function and mechanisms of receptor-interacting protein kinase (RIPK)-1, a protein that has been implicated in RIPK3-dependent and -independent inflammation and cell death signaling, including necroptosis. While the authors deliver an impressive study detailing the pathology of a number of KO and double KO models created through traditional breeding strategies, they utilized CRISPR guide sequences and Cas9 mRNA to generate a completely new transgenic model that targets the mixed lineage kinase domain-like protein (MLKL) gene in a keratinocyte specific RIPK1 KO mouse model (RIPK1E-KO). MLKL is a pseudokinase that is activated by RIPK3 and leads to TNF-induced necroptosis.
Dannappel et al. found that RIPK1E-K mice develop inflammatory skin lesions by one week of age, however deletion of MLKL prevented this phenotype. This indicates that RIPK1 is a regulator of epidermal homeostasis and suggests that necroptosis is a potent trigger for skin inflammation. Further conclusions from this study revealed a kinase-independent function for RIPK1 in epithelial apoptosis and necrosis and tissue homeostasis of the intestine.
The use of Cas9 mRNA has been gaining momentum over other methods, such as Cas9 plasmids. The transient nature of mRNA limits the expression of the Cas9 protein and allows the body to metabolize the mRNA quickly and naturally.
TriLink offers high quality Cas9 mRNA and Cas9 Nickase mRNA as well as reporter genes, such as EGFP and FLuc mRNA. Custom mRNA synthesis is also available from µg to gm scales.
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