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Genetic Engineering: an Overview of The Dna/rna and The Crispr/cas9 Technology

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As the field of Biotechnology grows, chemists and biologists alike are facing an ever-increasing conundrum of ethical hurdles brought on by technological breakthroughs. Genetic engineering stands on the cusp of a revolution. Breakthroughs in genetic engineering (GE) technologies will soon make it easier, cheaper, and more effective than ever before to alter the DNA of living organisms. GE technology is used to alter the DNA of living organisms, and has a number of different applications. One of the most promising applications of GE technologies is in gene therapy. Gene therapy is a technique where biologists alter the DNA of living subjects in an effort to cure genetic ailments (Gura, 2001). Early controversy over the use of gene therapy to treat disease was galvanized over the death of a hemophilic patient in a gene therapy trial (Gura, 2001). The greatest hurdle faced by any emerging GE technology is the uncertainty of the risks associated with it. The foremost GE technology today, CRISPR/Cas9, is no exception to this rule. Although CRISPR/Cas9 is set to revolutionize the world of genetic engineering there is considerable pushback from the general scientific community over the use of CRISPR/Cas9 and the potential side effects that could unknowingly stem from its use (Gilles & Averof, 2014). A brief overview of DNA/RNA, as well as CRISPR/Cas9 technology, coupled an analysis of the potential drawbacks and benefits of CRISPR/Cas9 will allow the reader to better understand the dynamics of the controversy and come to their own consensus on the matter.

To understand the mechanism of CRISPR/Cas9 one must be familiar with the function and process of DNA and RNA within the cell. DNA, deoxyribonucleic acid, is the building block of all life. It consists of two chains of complementary nucleotides. Each nucleotide consists of a phosphate group (PO4), deoxyribose sugar arranged as a ring of 5C (C5H10O4), and a nitrogenous base (Adenine, Guanine, Thymine, Cytosine). Nucleotides are covalently bonded to one another by a phosphodiester bond formed between the 5C sugar of one nucleotide and the phosphate group of another nucleotide. The two strands of nucleotides are bonded by hydrogen bonds formed between complementary nitrogenous bases. Adenine has complementarity for Thymine, whilst Guanine has complementarity for Cytosine (Thieman & Palladino, 2013). The two strands of nucleotides form a double stranded, double-helix structure referred to as DNA. DNA is essential to cell function as it codes for all proteins found in a cell. In order to produce a protein DNA must first be copied by another nucleic acid, RNA. RNA then sends a copy of the DNA to ribosomes in the cell which translates the sequence of RNA to an amino acid chain to form a protein (Thieman & Palladino, 2013). The entire process of transcription and translation is not within the scope of this paper, as such one only needs to understand the basic underlying function of DNA as a “code” for life, and RNA as a translator for DNA. DNA sequences are “read” as letters indicating the nitrogenous bases following the 5-prime (5’) to 3-prime (3’) direction. 5’ refers to the end of the sequence with a phosphate group bound to the fifth carbon of the sugar deoxyribose. For example, a sequence of Adenine-Thymine-Guanine-Cytosine, would read as ATGC. A particular sequence of DNA with a known function is referred to as a gene. The location of a gene is referred to as its locus.

Clustered regularly interspaced short palindromic repeats, or CRISPR, derives from a natural immune response of bacteria to viral infection (Gilles & Averof, 2014). CRISPR refers to a locus or loci found within the genome of bacterial cells. The mechanism of CRISPR involves incorporation of viral DNA to the CRISPR sequence in order to allow the bacteria to produce a strand of RNA that is complementary to the viral DNA. This is referred to as “CRISPR-derived RNA (Gilles & Averof, 2014)” or crRNA. The crRNA binds to “CRISPR-associated (Cas) proteins to form an active CRISPR/Cas endonuclease complex (Gilles & Averof, 2014).” An endonuclease is a protein that has the ability to degrade DNA. CRISPR/Cas9 refers to a specific endonuclease produced by the Streptococcus pyogenes bacterium. CRISPR/Cas9 contains two forms of RNA and the Cas9 protein. The crRNA contains the sequence necessary to complementary bind to the viral DNA. Another type of RNA, “trans-acting antisense RNA, also known as tracRNA”, contains the sequence necessary to “form a complex with Cas9 (Gilles & Averof, 2014).” Together crRNA and tracRNA form the “guide RNA” of the complex. The final piece, Cas9, is a protein that acts as the nuclease in the complex. The mechanism of CRISPR/Cas9 requires that a short sequence of nucleotides following the sequence targeted by the guide RNA (gRNA) must be present in the target DNA. This sequence, called a “protospacer adjacent motif” (PAM), is required for the function of Cas9 (Gilles & Averof, 2014).

The CRISPR/Cas9 complex has been modified by scientists to contain any desired gRNA sequence, which allows the targeting of any gene that contains the PAM. In bacteria, the CRISPR process ends with the even cleaving of DNA occurring a few nucleotides upstream of the PAM. For bacteria this is an effective method to destroy viral DNA. In eukaryotes, however, CRISPR/Cas9 is used to target a gene and insert, remove, or otherwise modify that gene. Engineered CRISPR/Cas9 complexes exploit two types of repair mechanisms used by DNA (Gilles & Averof, 2014). The first process non-homologous end joining or NHEJ, does not require a homologous strand (DNA containing the same genes with potentially differing alleles) of DNA for repair. In NHEJ the cut ends of the DNA are simply rejoined and the bonds are reformed. NHEJ may result in deletion of sequences of DNA, or it may introduce (insert) new DNA into the strand during repair (Reis, Hornblower & Tzertzinis, 2014). The other repair mechanism, homology-directed repair or HDR, requires a homologous strand of DNA in order to copy a short section of DNA used to repair the broken strand. HDR can be exploited by introducing homologous DNA containing a mutated or normal form of the gene being repaired. Removing a gene is referred to as knock-out and is usually done via NHEJ, and inserting a gene is referred to as “knock-in” and can be performed by NHEJ or HDR (Reis, Hornblower & Tzertzinis, 2014).

CRISPR/Cas9 confers a number of benefits over other gene editing technology. First, CRISPR/Cas9 is much easier than previously implemented gene-editing technologies. Two other technologies that work on a similar principle to CRISPR/Cas9, Zinc-finger nucleases and TALENs (Transcription activator-like effector nucleases), are much more technically difficult. Another benefit CRISPR/Cas9 offers is specificity, as long as the gene being targeted has the correct protospacer adjacent motif then CRISPR/Cas9 can be configured to target that gene. Finally an advantage CRISPR/Cas9 offers over TALENs is that it is not sensitive to methylation. Methylation inhibits the function of TALENs whilst it appears to have no effect on the function of CRISPR/Cas9 (Gilles & Averof, 2014).

The main disadvantage of CRISPR/Cas9 is the potential for off-target effects. Because CRISPR/Cas9 can tolerate differences of “up to 5 base mismatches within the protospacer region or a single base difference in the PAM sequence (Reis, Hornblower & Tzertzinis, 2014)” there is an opportunity to create “off-target mutations.” That is to say that the incorrect gene may be targeted as a result of very few differences in the nucleotide sequence of the target gene. Risks can be mitigated through careful experimental design, though the chance for off-targets cannot fully be eliminated (Gilles & Averof, 2014).

Concerns over the use of CRISPR/Cas9 stem from the many different applications of CRISPR/Cas9. Huge controversy was sparked when a team of Chinese scientists announced that they had “edited a gene in fertilized human eggs (Saey, 2015).” Much of the debate over the altering of human embryos is centered on the implications of allowing any modification of the human germline. As Saey notes, “critics worry that allowing genetic engineering to correct disease in germline tissues could pave the way to creating designer babies or other abuses that persist forever.” The experiment conducted by the team of Chinese scientists also highlighted the off-targeting problem associated with CRISPR/Cas9. Out of 86 total embryos only four were viable after CRISPR/Cas9 gene editing (Saey, 2015). Another concern with the use of CRISPR/Cas9, is the ecological effects of using the technique of gene drive to quickly and widely alter the genome of various flora and fauna (Ledford, 2015). Author Heidi Ledford explains that “researchers are deeply worried that altering an entire population… …could have drastic and unknown consequences for an ecosystem.”

The advantages of CRISPR/Cas9 make it a fantastic candidate for any number of gene-editing applications, though it is not without its drawbacks. The greatest problem facing CRISPR/Cas9 is in the novelty of its discovery and implementation. The debate over altering the human germline has existed long before the discovery of CRISPR/Cas9 and is likely to continue well into the current century. Most arguments offer a level headed approach to all issues associated with CRISPR/Cas9, the simplest solution is a modicum of caution. A great deal of concern comes from the fact that CRISPR/Cas9 is so revolutionary. The cost, effectiveness, and ease of use of CRISPR/Cas9 makes gene editing accessible to a far larger community than ever before. The best approach to dealing with CRISPR/Cas9 is simply more research focused on CRISPR/Cas9. Given the popularity of CRISPR/Cas9, this is most certainly the case for biologists today.

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Genetic Engineering: An Overview of the DNA/RNA and the CRISPR/Cas9 Technology. (2018, Jun 14). GradesFixer. Retrieved October 6, 2022, from
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