The CRISPR Story

From fixing neurological diseases like Alzheimer's, to creating designer babies and engineering mosquitoes to end malaria, advances in genome-editing technologies such as CRISPR–Cas9 have shown how rapidly the industry is moving. Gráinne Byrne tells you the nitty-gritty of how it works, and how it compares to alternative gene-editing technologies.

From Recombinant DNA Technology to CRISPR

The revolutionary development of recombinant DNA technology in the early 1970’s was a turning point in molecular biology, as the first successful direct transfer of DNA from one organism to another was achieved by Herbert Boyer and Stanley Cohen. Since then, research has advanced greatly, and more recent discoveries and innovations have completely revolutionised the way in which we are able to conduct genetic modifications. Instead of extracting DNA from one organism, isolating, altering and then re-inserting it into another organism, the function of DNA sequences can now be directly edited or modulated in their endogenous context.

Recombinant DNA technology has depended upon restriction sites called palindromic repeats, which occur approximately once in every 64,000 base pairs. This has proved to be a limitation of accuracy with regards inputting DNA to an exact location in the genome.

However, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique has overcome this barrier to success by using the endonuclease Cas9 (CRISPR associated protein 9) to splice DNA. This allows for the insertion of a purposely designed sequence which orders an sg-RNA (single-guide RNA) to match, and pieces of DNA can then be cut or pasted at virtually any specific location the biologist desires.

The first report of these palindromic clustered repeats was in 1987, but the acronym CRISPR was only coined in 2000 to bring under one heading the characterization of 'microbial genomic loci consisting of an interspaced repeat' (Zhang, Hsu). In 2002, the key CRISPR-associated proteins were identified.

CRISPR versus TALENs and ZFNs

Although CRISPR appears to be a breakthrough in gene-editing tool technology, it is not the first method which involved the utilisation of a nuclease for DNA snipping and precise modifications at the base pair protein level. The majority of the techniques operate by firstly placing a double-stranded DNA break at a specific location, which is then mended by the cell.

All techniques can be used for many applications, including gene knockout, transgene knock-in, gene tagging, and correction of genetic defects. The dividing line between the techniques are the way in which they introduce the break, and the level of difficulty in targeting new sequences. The most prominent alternative genomic-editing techniques to CRISPR are Zinc-Finger Nucleases (ZFNs) and Transcription Activator-like Effector Nucleases (TALENs).

DNA consists of an extended line of the 4 nucleotide base pairs (codons); A, T, C and G and CRISPR’s Cas9 can identify and recognise about 20 base pairs in a sequence. ZFNs and TALENs have the upper hand on CRISPR technology in how they can recognise longer sequences and be more suitably adjusted for a specific gene. However, such benefits come with a higher price as scientists have to compose the correct ZFN/TALEN protein, which usually takes more than one attempt to get right.

Although CRISPR directed sgRNAs can tolerate up to five mismatches with unwanted target sites (Fu, et al., 2013), CRISPR demonstrates a higher rate of efficiency in that an sgRNA can be created with more ease, and is more likely to work, all ultimately at a lower cost. (Zhang)