Background:
Since the discovery of DNA in 1953 by Watson and Crick, there has been rapid advancement in our knowledge of biological systems. In 1975 the Sanger Method, also known as the Chain Termination Method, was modified to rapidly sequence genomes and the method of shotgun sequencing, derived from these principles, was used in 1990 in the Human Genome Project. The aim of this project was to decode the entire 3,000 million bases (Mb) of DNA sequence and to interpret the information within it to better understand human genetics (Bentley, 2000). However, it is only in the last few years since CRISPR technology was discovered that truly precise and efficient genome editing has been able to occur in living eukaryotic cells (Lander, 2016).
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Structure and function of a CRISPR loci:
CRISPR within nature is effectively an adaptive immune system found in about 50% of bacteria and 90% of archaea to defend against viral infections (Wright, Nuñez and Doudna, 2016). It’s full name, clustered regularly interspaced short palindromic repeats, refers to its loci where CRISPR-associated Cas genes are located along with a series of direct repeat sequences and variable spacers: sequences which correspond to foreign genetic elements (Hsu, Lander and Zhang, 2014).
CRISPR was discovered after research emerged which showed that 88 out of 4,500 spacers from a range of bacteria and archaea were related to known sequences of bacteriophage and plasmids (Mojica et al., 2005). The hypothesis based on this was that the CRISPR machinery selected sequences from the phage genome after a previous invasion, and incorporated these as novel spacers to act as a memory library for future attacks. Later research proved that bacteria containing these spacer elements were immune to the corresponding foreign invaders whereas a closely related CRISPR-negative species were susceptible, thus acting as evidence for this hypothesis (Hsu, Lander and Zhang, 2014). Scientists were interested in how this incorporation of foreign genetic material could lead to an immune response, and discovered that these elements could be transcribed and processed into a set of small RNA’s (crRNA’s) that help direct the nucleolytic activity of Cas enzymes to degrade foreign nucleic acids (Karginov and Hannon, 2010).
This process as described in (Wright, Nuñez and Doudna, 2016), can be simplified into 3 stages and is also presented in figure 1:
1. Spacer acquisition:
Foreign DNA is identified, processed by Cas proteins, and finally integrated into the CRISPR locus as a new spacer between a series of direct repeat sequences.
2. crRNA biogenesis:
The spacer is transcribed as a single pre-crRNA which is processed into mature crRNA, each containing a single spacer.
3. Interference:
Finally, the effector complex (Cas enzymes) use the crRNA as a guide to identify and destroy phage or plasmids with sequences known as protospacers, sequences complementary to the guide spacer sequence of the crRNA.
Applications of CRISPR:
Although naturally the CRISPR mechanism has evolved for antiviral defence, it is the ease at which it can be customised that is of such interest and excitement. The crRNA sequence that guides the Cas enzymes can be replaced by a sequence of interest to retarget the Cas9 nuclease to different locations in the genome (Hsu, Lander and Zhang, 2014), which has huge implications for its use in basic research, biotechnology and medicine.
The most obvious of uses for the Cas9 endonuclease is in the precise inactivation of genes to see the effect of knock outs in a biological model. The accuracy at which the Cas enzyme cuts the complementary base pairs is unprecedented, based on past mechanisms for genome editing and manipulation such as ZFS and TALES. Once it was discovered that this system could be developed into a viable genome editing system, there was an explosion in the research to harness Cas9 and CRISPR (Hsu, Lander and Zhang, 2014). So far three variants of the Cas9 enzyme have been used in genome-editing processes, with two of these aimed to cleave DNA target sequences. The first example is wild type Cas9 aimed at cleaving double-stranded DNA, after which double-strand break (DSB) repair machinery repairs the DNA via a Non-Homologous End Joining (NHEJ) pathway (Gong et al., 2005). This pathway results in either insertions or deletions (indels), both of which disrupt the targeted locus and could be used to turn off genes.
The second edited form of Cas9, referred to as SpCas9, was developed by Cong and colleagues (Cong et al., 2013) and cleaves only one DNA strand. This means the NHEJ repair pathway is not activated reducing the number of indels in the disrupted target and, instead, DNA repairs from this nickase activity is performed only by the homology-directed repair (HDR) pathway, resulting in less indels. In fact, in the study by Cong, sequencing of 327 amplicons did not detect any indels induced by ScCas9 meaning this could be a better tool for precise genome editing in cases where no mutations are intended. Furthermore, the activity of the enzyme can work in a pair to cause adjacent nicks in order to create space for genomic insertions of the repair template (Cong et al., 2013).
Finally, the third version is a Cas9 is actually a nuclease-deficient version in which mutations in the HNH domain and other domains inactivate the ability of Cas9 to cleave but don’t affect its binding to DNA (Gasiunas et al., 2012). This version can be used to target any of the region without cleavage which makes it useful as a visualization tool (plagarsism). An example of this comes from Chen and colleagues (Chen et al., 2013) who used this nuclease-deficient Cas9 in conjunction with Enhanced Green Fluorescent Protein (EGFP) to visualise specific DNA sequences matching the targeted guide RNA in human cells. On the other hand, there have been issues raised with how accurate this visualisation tool can be considering that the Cas9 enzyme can occasionally associate with DNA similar, but not exactly the same, as the sequence guide RNA.
Specific example 1: Gene drive in mosquitos:
As mentioned previously, CRISPR can be used to insert new genetic material between a double stranded break, resulting in huge advances for many different fields of technology such as that involved with gene drives. These gene drives work by increasing the chance of their own inheritance in the next generation, therefore stimulating the spread of a modified gene throughout an entire population (Montenegro, 2016). Gene drives have long been thought of as a way to control and possibly exterminate the transfer of insectborne disease such as malaria, yellow fever and even the recently renowned Zika Virus. However, this technology has only been made a reality with the introduction of CRISPR as an editing system. It was in November 2015 that scientists at the University of California, Irvine, finally designed a working “selfish” gene capable of passing through mosquitos in a non-mendelian fashion (Regalado, 2015). Working in Anthony James’s basement insectary at the University, scientists were able to form a progeny of mosquitos with 99% red eyes from a mother with normal eyes and a father with red eyes. What was more revolutionary however, was the fact that those offspring with red eyes also carried the “selfish” gene to prevent the malarial parasite from growing.
The first addition in these CRISPR modified mosquitos is the introduction of genes that produce antibodies, which bind to the parasite’s (plasmodium) surface and stop its development, in response to the female mosquito having a blood meal. However as explained in this article, normally these genes would only be passed to only half of the next generation due to random assortment and segregation of chromosomes (Regalado, 2015). This is where the CRISPR comes in, introducing gene drive constructs (homing endonuclease genes called HEGs) designed to target and edit three genes (AGAP005958, AGAP011377 and AGAP007280) which confer a female-sterility phenotype upon disruption (Hammond et al., 2015). Proteins encoded with HEGs can recognise and cleave a specific 15-30bp DNA sequence which is then repaired using the opposite homologous chromosome as a template. This effectively turns a heterozygote into a homozygote, rapidly increasing the frequency of an HEG and associated genes in a population and, despite the cost of the disruption of the targeted sites, the benefit of the increased transmission rate could outweigh any negatives. In conclusion, if this gene drive system could be perfected and released into the wild, then CRISPR-edited insects could help reduce such deadly diseases such as Malaria which currently kills 670,000 people a year (Regalado, 2015).
Specific example 2: CRISPR/Cas9-mediated potyvirus resistance in Arabidopsis plants:
To simplify procedures in plants, the repeat sequences (crRNA) and protospacer of the CRISPR-Cas9 system can be combined into a single guide RNA (sgRNA) molecule which still acts as a guide to introduce DSBs. These DSBs, as previously mentioned, are repaired by the error-prone non-homologous end-joining (NHEJ) repair pathway. This means that Cas9-induced DSBs will often result in short indels at the site of DNA cleavage resulting in the inactivation of certain targeted genes (Pyott, Sheehan and Molnar, 2016).
In this study, the aim was to generate virus-resistant plants by inserting a loss-of-function mutation at the eIF9(iso)4E locus in Arabidopsis thaliana, which had previously shown to be associated with resistance to several potyviruses (Pyott, Sheehan and Molnar, 2016). The sgRNA was designed to be complementary to bases at the 5’ region of the open reading frame of the gene in question as this would increase the likelihood of translation into a non-functional protein by the addition of an early stop codon or by causing a frameshift. To test whether this method would work, the potyvirus Turnip mosaic virus (TuMV) was used and the resistance of wildtype and CRISPR edited plant samples from N.benthamiana was tested (Pyott, Sheehan and Molnar, 2016). TuMV was also modified to express a green fluorescent (GFP) in order to view presence of the potyvirus. Results such as those in Figure 2 showed that after inoculation for five days, TuMV-GFP infection was clearly visible from the wild-type plants whereas TuMV-GFP was not detected in the sap from any of the eIF(iso)4E mutants. This concludes that the use of the CRISPR-Cas9 system was effective at rendering the plants completely resistant to the TuMV infection.
It is clear that CRISPR has changed the future of genome editing by not only increasing the precision that genes can be edited, but also due to the fact that its simplicity and inexpensive nature opens up projects to labs all around the world. Many scientists support the use of CRISPR due to the speed at which it can be used to rewrite an organism’s DNA and its potential use in immunology, crop resistance and wiping out pathogens as mentioned in the malaria example.
However, there are also concerns about its use because such modifications, if done in gametes or a one celled embryo, will become permanent and fixed within the germ-line. A group of US scientists, including the co-developer of CRISPR Jennifer Douda, have stated that steps should be taken to “strongly discourage” attempts at this so called germ-line modification in humans before a meeting has been taken place to discuss ethical issues surrounding the use of this level of genome editing. Many believe there should be a discussion about what could, and should be achieved (The Guardian, 2015).
Despite these warnings, there are some groups that have begun to progress along the path towards permanent modifications in humans. In fact, a group of Chinese scientists are on the verge of taking the technology to the next level by planning to use the CRISPR-Cas9 system in humans to treat lung cancer. The team led by Lu You at Sichun University’s West China Hospital have succeeded in gaining approval from the hospitals review board but still need approval from other regulatory bodies. The team hope to use CRISPR to knock out a gene for PD-1 in T-cells, and use guide RNA to allow the T cells to home in on the cancer (Cyranoski, 2016). This indicates the massive potential benefits of continued research into gene editing systems especially when clinical trials will be carefully selected to only test on patients where chemotherapy and other treatments have failed. This use of CRISPR as a last resort and the regulation of CRISPR to only edit genes in somatic cells may persuade some who disagree with the ethics behind gene editing, but there is a long way to go before germ-line modification is accepted in the wider scientific community.