Genome Editing With CRISPR: Easy Targeting of Multiple Genes

Genome editing techniques have been extensively explored as methods to enable efficient and specific DNA cleavage that can be followed by homologous recombination (HR) between the host chromosome and a repair template. The advantage of genome editing over other techniques where modifications are carried out at the RNA/protein level or by introduction of transgene cassettes, is its persistence in both the modified cell and its progeny in a more relevant physiological context.

The concept of using engineered nucleases for genome editing is not new, but the description of a bacterial RNA-guided nuclease (Cas9) in 2012 and its application in genome engineering caused a stir in the scientific community because of its superior efficiency and ease of use. The method called clustered regularly-interspaced short palindromic repeats (CRISPR)-mediated gene editing, requires two components: a Cas9 nuclease and a guide RNA (gRNA) of approximately 20 nucleotides long. Based on the homology of the guide RNA and the host chromosome, Cas9 creates a double-strand break in the DNA.

The efficiency and specificity of the CRISPR/Cas9 system is promising for gene function studies and generation of transgenic models. Sakuma et al. explored the feasibility of multiplex genome engineering in human cells by delivering multiple gRNA molecules simultaneously together with the Cas9 nuclease. Using an all-in-one system they demonstrated that up to seven gRNAs could be delivered in one plasmid together with Cas9 and induce chromosomal editing at their corresponding target genes. The ability to perform simultaneous gene editing of up to seven loci in one experiment is of extreme value as it saves researchers costs and time when studying complicated pathways where the function of a number of genes needs to be studied in relation to other pathway components. In addition to demonstrating the feasibility of multiple genome editing events in one go, the researchers also compared the efficiencies of multiplexing to individual targeting of each locus and found them to be very similar.

As with all genome editing techniques, off-targeting is of equal importance to DNA cleavage efficiency since promiscuous activity of any given nuclease could introduce unwanted mutations in the host’s genome. The CRISPR/Cas9 system is also prone to off-targeting and mutated versions of Cas9 exist that transform it into a nickase requiring two gRNAs per target in order to increase target recognition specificity and reduce off-targeting to nearly undetectable levels. In the current study, multiplexing was also shown to work with the Cas9 nickase for the simultaneous targeting of three chromosomal loci.

Genome editing is likely to be at the forefront of future studies on gene function and, indeed, novel therapeutics. Further optimisation and characterisation of the CRISPR/Cas9 system is currently under way by multiple research groups worldwide. This paper tests the efficacy of the technique at its current maximum and it’s surely one of many to follow in the search for a method to elicit efficient, fast and safe genome editing.

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References

Jinek M., K. Chylinski, et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science 337(6096):816-21

Sakuma T., A. Nishikawa, et al. (2014). Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system Scientific Reports 4:5400

 

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