This was written in 2018 as part of course on being green and renewable energy sources in modern times.
A new revolutionary genome-editing tool called CRISPR has been and continues to change the landscape of genetics and bioengineering. In borrowing this selective and programmable bacteria/archaea defense system, scientists have been able to do things such as: prevent heart disease at the level of the human embryo and develop test strips for field medical diagnostics. As a result of these achievements and the versatility of CRISPR, general interest from the public has been sparked and has lead to much speculation of a genetically engineered future. There is no doubt that genetic and bio- engineering is being and has been revolutionized by this new technology, however some border cases of its uses are more fiction than fact.
Clustered regularly interspaced short palindromic repeats (CRISPR) is the name for the part of a genome responsible for storing the genetic information of invading bacteriophages. It is an evolved, adaptive defnese system used by bacteria and archaea [1]. The system relies on small RNAs for sequence-specific detection and silencing of foreign nucleic acids [1]. The system is composed of cas genes organized in operons made up of genome-targeting sequences called spacers which are interspaced in regula intervals between palindromic repeats [1]. The CRISPR Cas system works by selectively recognizing and then cutting or silencing specific nucleic acid sequences. A guide RNA (gRNA) a transfer RNA (transRNA) and a target sequence are used by the cas protein to find a complementary strand of RNA to the target sequence. Once the target sequence and a particular protospacer adjacent motif (PAM) is found, the protein binds and then does something depending on how the protein was matured or mutated [2]. In its wildtype form, the Cas9 protein will cut at the region on both sides of the DNA, at the approximately the same length, or the single side of the RNA [3]. There a three classes of
CRISPR/Cas systems. Types 1 and 3 are similar in that the cas protein endonucleaes process the pre-CRISPR RNA (pre-crRNA), develop into a full protein, and cleave RNA complementary to this RNA. Type 2 contrasts in that is developed using a double-stranded (ds) RNA-specific ribonucleae in the presence of Cas9 and this protein is necessary in the crRNA-guided silencing of foreign DNA [1]. Similarly, Cas13 is necessary in processing of foreign RNA [4]. Given that the CRISPR/Cas system only needs to have the DNA sequence of interest in its library, it is highly programmable and easily employed. It is for this reason that CRISPR is so promising a technique. Moreover, it has captured the attention and imagination of people in the general population, with the publication of books like Prometheus [5], academics, politics, and business, with various bioengineering based startups [6]. CRISPR has potential to be used to make edits anywhere nucleic material is used, e.g. animal embryos, plant seeds, parasites, viruses, etc and so its uses vast. Also, it allows for the ease of detection of such material due to Cas9/13’s programmability and selectivity [6].
It was demonstrated by Cox et. al, that the CRISPR-Cas13 system could make RNA edits with surgical precision. A RNA-editing reporter on a Cluc was introduced by a point mutation at a single nucleotide changing the guanine nucleotide to an adenosine nucleotide. This nonsense mutation could be functionally repaired to its wild type codon by mutating the A to an I by use of dCas13b-ADARDD(E488Q) [3]. This system, called RNA Editing for Programmable A to I Replacement version 1 (REPAIRv1), validated that precises RNA editing could be achieved at at test site. It was also noted that this editing occurs more accurately with longer (50 nucleotide spacers) as opposed to shorter (30 nucleotide-spacers) spacers, however with longer spacers around the target site, there was an increased propensity for editing at non-targeted adenosines within the a given sequencing window [3]. Furthermore, the REPAIRv1 system was demonstrated to work on living mammalian cells in order to correct human disease related to mutations [3].
It was demonstrated by Abudayyeh et. al, that RNA-guided RNA-targeting through the CRISPR–Cas13 system could be engineered for mammalian cell RNA knockdown and binding. Cas13a specificity was tested in vivo by introduction of single mismatches into a guide targeting either Gluc, a reporter construct, or endogenous genes. It was found that the knockdown was sensitive to mis- matches in the central seed region of the guide-target duplex, which was additionally confirmed by biochemical profiling. Given that RNA interference is known for having off-target affects, a transcriptome wide mRNA sequence was run in order to assess for off-target affects. This was done by providing the Cas13 with an option of either the target region or shRNA construct and it was found that significant knockdown of the target transcript (P < 0.01) [4].
A stochastic model of gene flow by gene drive was developed in order to understand how gene drive could lead to fixation. The underlying assumptions of the model are finite population and that it is based off of a Moran-based model. It was found, through numerical simulation of subpopulations and 3 alleles, wild type, gene drive, resistance, on diploid organisms, that isolation, as a prevention of invasion from gene driven populations of a species, unless migration rate is kept extremely low (10−5 on a scale of 0 to 1), that gene flow is almost guaranteed in invade all sub-populations linked to the originating one leading to fixation of the gene drive [7]. That is, CRISPR gene drive systems capable of far rearching spreads unless heavly isolated.
CRISPR’s ease of use, quickness of application, and inexpensiveness is why it is such an exciting technology. In particular, the potential to genetically modify human embryos in vivio has attracted the attention of the general public for its ethical and genetic ramifications [8]. Questions how would such a technology be regulated, how much would it cost, and who would have access to it are immediate question to the possibility of this embryonic editing being commercialized. [5] Additionally, CRISPR’s potential to optimize organisms could lead to highly invasive species in wild populations [7]. Nevertheless, the reality of CRISPR is that it is currently being used to quickly test for the nucleic material of potentially harmful things [9].
A lab at MIT developed a way to use CRISPR for to rapidly and inexpensively detect nucleic acid material. The diagnostic tool SHERLOCK (Specific High Sensitivity Reporter unLOCKing) is a paper strip display test the uses a fluorescent RNA reporter to report whether or not a specific nucleic acid material is present in a medium [6]. If the nuclear material is present, Cas13 will bind to the material and cut it, but due to the nature of the enzyme, it will bind to and cut the reporter as well as other nearby nuclear material. This side-effect was taken advantage of and was used to cause a test strip to display a black line, similar to a pregnancy test, in minutes. This rapidity and ease of use gives it the potential to change public health in areas such as Africa and South America or even in hospitals for the detection of tumor cells [9].
Genetic editing using the CRISPR/Cas system has been used in both China and the United States to edit the genetics of embryos. In the United States, CRISPR-Cas9 has been used to correct a genetic defect that can lead to heart failure [10]. As the change was made to the diploid formed after sperm met the egg, the change is heritable. The news of being able to do this has sparked a lot of ethical debates about the use of human germline editing to alter humans to have anything from blonde hair to wings. Despite the power of CRISPR, this is unlikely to occur. The idea for this rests in the adage of nature vs. nurture and that a child’s talents and abilities are said to fall more so in the latter category than the former. Additionally, even the case of that we could alter human beings at the level of talent from the level of genetics, the off-target effects or whether the resulting embryo would develop are speculative [8]. Literature demonstrates changes at a single locus and more so at a single or a few nucleotides, not at multiple loci for large numbers of nucleotides.
Another potential use of CRISPR is in the bioengineering of gene drives. Gene drives are genetic advantages programmed to specific alleles such that if one copy of the allele is present, it is passed on to the off spring, in sexually reproducing organisms, with a much higher than 50% probability [11]. This phenomonea is not new and has been observed in fruit flies since the 1950s [11], however, the idea of using it as a tool to control or eliminate the spread of certain diseases is new and point of contention. There are arguments for the use of gene drives arguing that it is beneficial for eliminated invasive species and pathogens like malaria and that in the case of of mixing with wild-type alleles, resistance at the locus would prevent drive fixation [12]. However, others argue that such a mechanism are likely to be highly invasive and can result in fixation of the chosen allele in a population [7].