Insights on CRISPR-Cas9 Genome Editing

Marcella Faria^*
*Correspondence: Marcella Faria, Department of Molecular Biology, University of Cologne, Germany, Email:

Introduction

CRISPR-Cas9 is a novel technology that allows geneticists and medical researchers to edit parts of the genome by removing, adding, or changing DNA sequences. It is currently the simplest, most versatile, and precise method of genetic manipulation and it is causing quite a stir in the scientific community. The CRISPR-Cas9 system is made up of two key molecules that introduce a change into the DNA. Is it an enzyme referred to as Cas9. This functions as a pair of 'molecular scissors' cutting the two strands of DNA at a specific location in the genome, allowing bits of DNA to be added or removed. A strand of RNA referred to as guide RNA (gRNA). This is made up of a small piece of pre-designed RNA sequence (about 20 bases long) that is embedded within a larger RNA scaffold.

The scaffold binds to DNA, and the pre-designed sequence 'guides' Cas9 to the correct region of the genome. This ensures that the Cas9 enzyme cuts at the correct location in the genome. The guide RNA [1-3] is programmed to locate and bind to a specific sequence of DNA. The RNA bases in the guide RNA are complementary to those in the target DNA sequence in the genome. This means that, in theory, the guide RNA will only bind to the target sequence and will not bind to any other regions of the genome.

About the Study

Some bacteria have a built-in gene editing system similar to the CRISPRCas9 system, which they use to respond to invading pathogens such as viruses. Similar to an immune system. The bacteria use CRISPR to snip out parts of the virus DNA and save a piece of it to help them recognize and defend against the virus the next time it attacks. Some bacteria have a builtin gene editing system similar to the CRISPR-Cas9 system, which they use to respond to invading pathogens such as viruses. Similar to an immune system the bacteria use CRISPR to snip out parts of the virus DNA and save a piece of it to help them recognise and defend against the virus the next time it attacks. This system was modified by scientists so that it could be used in other animal cells, including mice and humans. CRISPR-Cas9 [4,5] has a lot of potential as a tool for treating a variety of genetically based medical conditions, such as cancer, hepatitis B, and even high cholesterol. Many of the proposed applications involve editing the genomes of somatic (non-reproductive) cells, but there has been considerable interest in and debate about the possibility of editing germline (reproductive) cells.

Because changes made in germline cells are passed down from generation to generation, they have significant ethical implications. Gene editing in germline cells is currently illegal in the United Kingdom and most other countries. The use of CRISPR-Cas9 and other gene editing technologies in somatic cells, on the other hand, is uncontroversial. They have already been used to treat human disease in a few exceptional and/or life-threatening cases. It will most likely be many years before CRISPR-Cas9 is routinely used in humans. Much research is still focused on its use in animal models or isolated human cells, with the goal of eventually using the technology to treat diseases in humans on a regular basis. There is a lot of work being done to eliminate 'offtarget' effects, which occur when the CRISPR-Cas9 system cuts at a different gene than the one intended to be edited. In most cases, the guide RNA is composed of a specific 20-base sequence.

Conclusion

These are complementary to the target sequence in the gene under consideration for editing. However, not all 20 bases must match for the guide RNA to bind. The problem with this is that a sequence containing, say, 19 of the 20 complementary bases may exist in a completely different location in the genome. This means that the guide RNA could bind there instead of or in addition to the target sequence. The Cas9 enzyme will then cut at the incorrect site, introducing a mutation in the incorrect location. While this mutation may have no effect on the individual, it could affect a critical gene or another important part of the genome.

References

1. Cheng, Albert W, Haoyi Wang and Hui Yang, et al. "Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system." Cell Res 23 (2013): 1163-1171.

Google Scholar, Crossref, Indexed at

2. Bassett, Andrew R, Charlotte Tibbit and Chris P. Ponting, et al. "Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system." Cell Rep 4 (2013): 220-228.

Google Scholar, Crossref, Indexed at

3. Rong, Zhili, Shengyun Zhu and Yang Xu, et al. "Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template." Prot Cell 5 (2014): 258-260.

Google Scholar, Crossref, Indexed at

4. Sander, Jeffry D and J. Keith Joung. "CRISPR-Cas systems for editing, regulating and targeting genomes." Nat Biotechnol 32 (2014): 347-355.

Google Scholar, Crossref, Indexed at

5. Bikard, David, Wenyan Jiang and Poulami Samai, et al. "Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system." Nucleic Acids Res 41 (2013): 7429-7437.

Google Scholar, Crossref, Indexed at