(Picture credit: Stephen Dixon and Feng Zhang via Wired)
An arms race has been waged between bacteria and bacteriophage that would bring a satisfactory tear to Sun Tzu’s eye. Scientists have recently recognized that countermeasures developed by bacteria (and archaea) in response to phage infections can be retooled for use within molecular biology. In 2013, large strides have been made to co-opt this system (specifically and most commonly from Streptococcus pyogenes) for use in mammalian cells. This countermeasure, CRISPR (clustered regularly interspaced short palindromic repeats), has brought about another successive wave of genome engineering initiated by recombineering and followed more recently by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs and TALENs perform a similar function yet the learning curve appears to be more difficult for development due to the use of protein-DNA contacts rather than the simplicity of designing RNA-DNA homology contacts. Although the potential for CRISPR in regards to genome editing within mammalian cells will be of greatest interest to the reader, the CRISPR backstory is equally compelling. Just as we have evolved immune responses to pathogens, so too have bacteria. CRISPR is an adapted immune response evolved by bacteria to create an immunological memory to ward off future phage infections. When a phage infects and injects its DNA within a bacterium, the DNA commandeers bacterial proteins and enzymes for use towards lytic or lysogenic phases. However, exposure of phage DNA allows the bacterium to copy and insert snippets (called spacers) of phage DNA into its genomic DNA between direct repeats (DR). These snippets can later be expressed as an operon (pre-CRISPR RNA, pre-crRNA) alongside a trans-activating CRISPR RNA (tracrRNA) and an effector CRISPR associated nuclease (Cas). Together these components surveil for foreign crRNA cognate sequence and cleave the targeted sequence. Although hallmarks of CRISPR have been known since the late 80’s (CRISPR timeline) and was acronymed in 2002, Jinek et al. in August 2012 were the first to suggest the suitability of CRISPR towards genome editing. In February of 2013, Feng Zhang’s and George Church’s labs simultaneously published the first papers describing the use long oligos/constructs for editing via CRISPR in mammalian cells and made their plasmids readily available on Addgene. Zhang’s lab went one step further and has supplemented their papers with a helpful website and user forum. They have even gone so far as to publish a methods paper to streamline the use of their plasmids towards a plug-and-play, modular cloning approach with your target sequence of interest.
CRISPR works fairly well out of the box yet still has some imperfections that are being addressed. For example, CRISPR relies upon a protospacer adjacent motif (PAM; S. pyogenes sequence: NGG) 3’ to the targeting sequence to permit digestion. Although the ubiquity of NGG within the genome may seem advantageous, it may be limiting in some regions. Other species make use of different PAM sites that can be considered when choosing a cut sites of interest. Since double-stranded cuts could potentially create DNA lesions (a byproduct of the cell using non-homologous end joining [NHEJ] instead of homologous recombination) some labs are choosing to use modified Cas enzymes that nick DNA, instead of creating a double-strand break. This potential weakness of CRISPR to create DNA lesions via NHEJ, however, has been exploited by Eric Lander’s and Zhang’s lab this month (Jan. 2014). They have capitalized on the cell’s use of NHEJ to manufacture DNA lesions (frameshift mutations) at cut sites within genes on a large scale as a means to perform large genetic screens. Using this technique knocks out a gene and has the obvious advantage of fully ablating a gene’s expression compared to RNAi where some residual expression can be expected.
The advantages of CRISPR lends itself to future therapies. High efficiency, low-to-no background mutagenesis and easy construction put CRISPR front and center as the tool de jour for gene therapy. In combination with induced pluripotent stem cells (iPSCs), one can imagine the creation of patient-specific iPSCs created with non-integrative iPSC vectors and modified by CRISPR, devoid of any residual DNA footprint left behind by the iPSC vector or CRISPR correction. In conjunction with whole genome sequencing, genetically clean cell lines can be selected that are suitable for differentiation towards the germ layer of interest for subsequent autologous transplantation. Proof of principle experiments have already been published in models of cystic fibrosis and cataracts.
For better or worse, CRISPR is catching on like wildfire with young investigators, as noted recently by Michael Eisen. What may be looming in the future and not as openly discussed at this time is the potential for CRISPR to open up the genome to large scale editing. We tend to think of any particular genome as fairly static with slight variations between any two individuals and increased variation down the evolutionary line. However, CRISPR has proven to be a fantastic multitasker, capable of modifying multiple loci in one fell swoop as demonstrated by the Jaenisch lab (five loci). With the creation of Caribou Biosciences and a surprising round of venture capital raised by a powerhouse team at Editas Medicine in November ($43 million), CRISPR appears to also have sparked an interest in the private sector. With large sums of money at their disposal, these companies can now begin to look at the genome, not as a static entity, but more akin to operating system, a code that now has a facile editing tool. George Church, an Editas co-founder, has speculated in the past about the potential use of the human genome as the backbone for recreating the Neanderthal genome in his recent book and interview with Der Spiegel. In an era where the J. Craig Venter Institute can create an organism’s genome de novo and a collaboration between Synthetic Genomics and Integrated DNA Technologies has proposed to synthesize DNA upwards of 2Mbp, the combination of CRISPR, synthetic DNA and some elbow grease will make the genome more accessible and Church’s speculations a potential reality.