The following is report on a new method of genetic manipulation. It was written for a work related task and therefore is more complicated and professional than my other posts. As such it lacks my usual sarcastic wit you’ve all come to love. If you are a casual reader you may want to skip this one. If you are a potential employer, enjoy.

An ancient immune system used by bacteria to combat against viral phage infections is the latest tool at the disposal of genetic engineers. Many have high hopes of it allowing not only targeted genome alterations, but also the ability to colocalize any RNA, DNA or protein polymer to specified genomic DNA locations. CRISPR (clustered regularly spaced short palindromes repeats), like its name suggests are regions of repetitive palindromic sequences that are separated by seemingly random short stretches of DNA. The clusters form a repeating palindrome-spacer-palindrome-spacer-etc stretch of DNA. The unusual clusters have been found in nearly half of bacteria and over 90% of archea microbes. Initially written off as junk DNA the secret to CRISPR’s functional role resided in the seemingly not so random spaces between its palindromes. In 2005 after closer examination by a number of bioinformatics teams, the sequence of these DNA “spacers” was shown to share similarity to a number of bacteriophages, viruses that infect microbes. Two years later a team at the food science corporation Danisco showed that manipulating the sequence of these spacers altered microbial immunity to phages with homologous DNA. Further research concluded that microbes saved pieces of invading bacteriophage DNA as spacer CRISPR regions to be used later in an adaptive RNAi like immunity system. The excitement of the microbiology community not withstanding, this newly defined system would soon be exploited by genetic engineers due to its sequence specific DNA targeting capabilities.

Much like RNAi, the immunity role of CRISPR relies not only on its DNA sequence to target invading viruses, but also on nuclease enzymes to destroy their foreign DNA. In bacteria, CRISPR loci are flanked by a large number of CRISPR associated (cas) genes that play multiple roles in the microbial CRISPR immune response. Investigators have stripped down the CRISPR-Cas system in efforts to transform it into a viable genetic engineering tool. A typical CRISPER-Cas experiment relies on two components: 1. An RNA that contains a small region of homology to the target gene termed short guide RNA (sgRNA) and 2. A nuclease called Cas9. Together these two components hone in on the region of the genome targeted by the sgRNA that is then cut via Cas9. The end result is a relatively simple and fast technique to mutate your gene of choice. So far the results of CRISPR-Cas-mediated experiments have been promising making it arguably the most exciting new methodology in genetic engineering. In less than a year it has been used to mutate endogenous genes in a number of organisms including fruit flies, mice and human cells. The mouse community is particularly optimistic due to the speed CRISPR-Cas offers over traditional engineering methods. CRISPR-Cas has been used to create mutant mouse lines in the span of weeks; a tremendous upgrade to classical methods, relying on homologous recombination, which can take up to a year.

The versatility of CRISPR-Cas goes beyond simply creating mutants. As noted by the Church lab, one of the pioneers in CRISPR studies; the CRISPR-Cas system is the first known example of “a programmable targeting system capable of colocalizing all three types of sequence defined biological polymers” (DNA, RNA and protein). By attaching transcriptional activators or repressors to a nuclease null version of Cas9, investigators have taken preliminary steps to regulate endogenous gene expression. Additionally a “milder” version Cas9 has been made to nick, not cut, DNA allowing incorporation of new exogenously supplied DNA through homology directed repair. This has led to the incorporation of reporter constructs, precise point mutations and loxP elements in mice to create conditional mutants.

Cell lines and model organisms such as fruit flies, zebrafish and mice are routinely used to study human diseases whose roots can often be traced to genetic mutations. The advent of next generation sequencing has given us a much better view of the genetics involved in various diseases. The amount of data in the form of genetic mutations has now surpassed our ability to properly model these multigenic diseases. Studying complex genetic diseases requires complex genetic models. To date, CRISPR-Cas has been used to simultaneously disrupt five genes in a mouse.  Imagine the advantage, for instance, of using a mouse cancer model with multiple endogenous oncogenes upregulated combined with conditional knockout of multiple tumor suppressors. Such a mouse would have huge advantages over current xenograft and simpler genetic mouse models. CRISPR-Cas stills has hurdles to overcome such as perfecting how its components are delivered into specific model organisms and, perhaps more importantly, controlling the rate of any unwanted off-target effects. However, these are typical expected issues considering how young the CRISPR-Cas revolution is. Calling CRISPR-Cas a revolution may be premature, but the speed at which large biotech companies have incorporated its tools into their catalogs and the startup of smaller companies devoted solely to its technology is proof that this system derived from “microbes with the flu” will get its shot.

Yang, H., Wang, H., Shivalila, C., Cheng, A., Shi, L., & Jaenisch, R. (2013). One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering Cell, 154 (6), 1370-1379 DOI: 10.1016/j.cell.2013.08.022