The immune system of vertebrates consists of two main strategies; innate immunity and adaptive immunity. The adaptive immune system relies on the ability to generate genetic variation at a cellular level through targeted somatic hypermutation, a mechanism by which the immune system develops antibodies capable of detecting and isolating specific foreign antigens. Somatic hypermutation is carried out by the enzyme ‘activation-induced cytidine deaminase’ (AID) which alters the variable regions of the antibody genes responsible for coding for the antigen binding domains of the antibody molecule.
Prokaryotes, such as bacteria and cyanobacteria, also display adaptive immunity through a different mechanism: ‘CRISPR’ (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated). This mechanism records and destroys foreign DNA and RNA by cutting and combining it into a host genomic region called the CRISPR-array. The CRISPR-array region is transcribed to produce crispr-RNA which then allows recognition of the complementary foreign DNA/RNA.
An adapted CRISPR/Cas9 system has recently been adopted by scientists as a gene editing tool for eukaryoke cells. In a recent study Nishida and colleagues have used nucleotide editing using a hybrid vertebrate-prokaryote system to investigate whether the CRISPR/Cas9 system may be used as a targeting tool to enable AID to carry out site specific mutations. In this study a mutant Cas9 from the bacteria Streptococcus pyogenes was combined with an enzyme with the same function as AID from the sea lamprey, to form a synthetic complex (Target-AID). This was expressed in the yeast Saccharomyces cerevisiae. The CAN1 gene (hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus) was targeted, as loss of function of this gene results in resistance to the drug canavinine.
To investigate the effectiveness and efficiency of this synthetic complex in mammalian cells; Chinese hamster ovary (CHO-K1) cells were transfected using a vector expressing Cas9 or the mutant Cas9 and RNA targeting the HPRT locus. The cells were grown in Ham’s F12 medium and mutation rates calculated by counting resistant colonies. In addition, off-target effects were assessed through whole genome sequencing.
The results demonstrated efficient site-specific mutations within a narrow range (3-5 base pairs of the target) in yeast and also resulted in deletions in mammalian cells. Toxicity was reduced and the off-target effects were similar to those of traditional CRISPR/Cas systems. The study therefore concludes that by combining the CRISPR/Cas9 system and a deaminase-mediated hypermutation (ie using Target-AID), a narrow range of effect is achievable for targeted nucleotide substitution which may, following further investigation, increase the capacity of genome editing.
34 CHO cell lines are available from ECACC, the majority of which are clonally-derived from the cell line CHO-K1 used in this study. The first CHO cell lines were developed by Theodore Puck in 1958 as tools for studying the effects of physical and chemical agents on mammalian chromosomes. In comparison to cell lines from other species, including humans, the CHO cell lines were chromosomally unstable, allowing the generation of a series of sub-clones. It soon became evident however, that CHO cell lines were capable of rapid growth to high cell densities in serum free conditions; they could be could be genetically manipulated with relative ease and capable of expressing secreted and membrane expressed proteins in high quantities making the cell lines valuable workhorses in the research, bioprocessing and drug discovery fields.
Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., . . . Kondo, A. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science (New York, N.Y.), 353(6305), Science (New York, N.Y.), 16 September 2016, Vol.353(6305).
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