Genomic Editing à la carte

The recent decades have witnessed what has been named as a Genomic Revolution. The most recent discovery in this revolution is called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (an RNA-guided endonuclease) system, a breakthrough new form of DNA editing. The system was originally discovered in bacteria and archae in the late 80’s. Microbiologists found in the genome of these organisms patterns of interspersed DNA whose function had remained elusive for many years. Several decades after, through sequencing of bacterial genomes, researchers discovered that these repeats were flanking DNA sequences of virus origin that the bacteria had incorporated into their chromosome. Moreover, these elements (CRISPR) were found to be in close proximity to genes that coded for proteins (Cas enzymes) involved in DNA cleavage and repair (Bolotin et al., Microbiology 2005; Mojica et al., J Mol Evol 2005; Pourcel at al., Microbiology 2005). Over the following years it was found that these viral sequences inserted at these specific loci constituted an immune memory that allowed bacteria fighting invading nucleic acids –such as virus- and blocking their propagation, and was the first evidence of an acquired immunity used by bacteria to adapt against foreign DNA. 

This mechanism of defense starts with the detection of foreign DNA that has been injected in the bacteria by a virus. This viral DNA is detected by the CRISPR system, integrated into the CRISPR locus, and is subsequently used to form RNA-interference (guide-RNAs) complexes that are able to base-pair with invading nucleic acids and induce their cleavage. Pioneering works by the groups of Emmanuel Charpentier and Jennifer Doudna (Wiedenheft, B., et al., Nature 2012; Jinek M et al., Science 2012; Doudna JA, Charpentier E, Science 2014) uncovered how bacteria had evolved to use their invaders and turn the sequence information against them, and managed to manipulate this system to create a method of programmed DNA editing. In 2013, the groups of Feng Zhang and George Church described how by manipulating the RNA molecules (single-guide RNAs; sgRNA) that, through pairing with specific endogenous genomic loci recruit the Cas9 endonuclease, it was possible to specifically and permanently stimulate the editing of the mammalian cellular genome (Mali, P. et al., Science 2012; Cong L, et al., Science 2013).

The principle of this assay is a sequential two-step process: (1) the Cas9 nuclease is guided by pairing guide-RNAs (sgRNA) to a specific sequence where the DNA cleavage occurs; (2) the double strand breaks on the DNA then activates the endogenous DNA-repair pathways. This DNA repair can occur through two major pathways: nonhomologous end joining (NHEJ), and homology-directed repair (HDR) pathways. The former results on the introduction of insertions/deletions (indels) at the target site, frequently resulting on loss-of-function; the later requires a homologous recombination (HR) involving the use of a similar or identical DNA molecule that is not broken and can provide a template to restore the code or to generate genetically “edited” variants.

Figure 1: Cellular DNA repair-pathways are activated in response to CRISPR/Cas9-mediated cleavage. Error prone NHEJ introduces random indels to reconstitute the DNA after strand breaks (left); HDR involves the use of a template that directs the addition of nucleotides to precisely reconstitute the code. (Image from F Ann Ran, et al, Nature Protocols 2013).

The tremendous versatility of this genome engineering technology holds great potential.  A series of molecular biology tools have been developed such as the introduction of GFP tags to study the cellular distribution of specific proteins within the cell; or to detect and capture specific sequences for downstream applications. On the industrial side, potential applications are the production of livestock, plants, and microorganisms carrying useful traits. The list of possibilities is far-reaching.  In biomedicine, it is being increasingly used to study the function of genes and their involvement in diseases, and holds promise to for the therapeutic correction of genetic defects in humans.  Recently published works have revealed how CRISPR technology can be used to correct genetic mutations in whole animals (Yoshimi, K et al., Nature Communications 2014; Heo, T. Y. et al., Stem Cells Dev 2015) and to direct the fate of cultured stem cells towards producing specific tissues in vitro (Z. Zhu, F. González, and D. Huangfu, Methods Enzymol 2014).  Besides, the implementation of sequencing protocols to uncover genetic alterations that influence the development of disease has exposed a scenario for potential intervention.

However, further research will be required to fully understand the mechanism and regulation of the CRISPR/Cas9 system, and especially on how the levels of Cas9 within a cell can generate off-target cleavage that result in unwanted side-effects such as genetic mosaicism or insertion of additional mutations.

A recent paper published by a group of Chinese scientists (Liang P et al, Protein Cell 2015) has reported for the first time the modification of human embryos using the CRISPR/Cas9 system, and have sparked the debate about what types of gene-editing research are ethical. On this work, scientists attempted to correct a genetic anomaly that is responsible for β-thalassemia, a heritable potentially fatal blood disorder characterized by mutations on the genes that mediate the synthesis of hemoglobin. The team injected 86 pre-implantation embryos with the CRISPR/Cas9, and found that only 4 of them contained the genetic material introduced to repair the defect. In addition, researchers also found several mutations that were consequence of off-target activity of the CRISPR/Cas9 activity. In summary, the results obtained by this study reveal the prospective use of this technology to potentially cure serious devastating diseases, but also prove the immaturity of the technique to be applied with therapeutic purposes, and raises safety and ethical concerns on human experimentation and germline editing.

Once again, science has proven to be ahead of legislation. It is therefore required that the scientific, medical, ethical and legislative policies evolve and adapt to regulate such a daunting revolution, especially on what concerns the modification of the germ line to enhance heritable human traits. Several committees of world-renowned experts are designing practical recommendations to channel genome editing research (Baltimore D et al., Science 2015). However, explicit legislation must be set in place to anticipate unwanted situations.

Today we are able to change the DNA à la carte and push scientific progress to a new edge; however, with great power comes great responsibility, and the scientific community needs to be cautious and aware of the consequences for the sake of the future days. 

Francisco Javier Carmona (BIO'07)

Research Fellow. Dr Baselga Lab.

Human Oncology & Pathogenesis Program

Memorial Sloan Kettering Cancer Center