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How Cas9 Detects and Cleaves DNA Targets, Essay Example

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Essay

How Cas9 detects and cleaves DNA targets and (eukaryotic) cellular responses to the resultant double strand DNA break

CRISPR-Cas9 systems have been quickly known scientific engineering tools due to their simplicity and great productivity. CRISPR-Cas9 comprises two main parts: the Cas9 enzyme and a guide RNA (gRNA) (Filippova et al. 2019). The gRNA binds to specific DNA sequences in the target’s genome and directs Cas9 to cleave those targets (Zhu, Holmes, Aronin and Brodsky 2014). This process results in a double-strand DNA break (DSB) which various mechanisms can then repair, including non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Schiermeyer et al. 2019). This essay will discuss how Cas9 detects and cleaves DNA targets and cellular responses to the resultant DSB.Cas9 is a DNA-targeting enzyme that is composed of two domains: the nuclease domain and the RuvC domain. The nuclease domain contains the Cas9 active site, cleaving DNA targets (Jiang et al. 2016). The RuvC domain binds to the gRNA and detects DNA targets (Shams et al. 2021). When the gRNA binds to the target DNA sequence, it forms a duplex with the target DNA. This causes the RuvC domain to bind to the gRNA-DNA duplex, which recruits the Cas9 nuclease domain to cleave the target DNA.

The specificity of the CRISPR-Cas9 system is due to the guide RNA (Prykhozhij et al. 2015). The guide RNA is designed to bind to a specific DNA sequence in the genome called a target (Qi et al., 2013). The gRNA contains a 20-nt sequence called the adjacent protospacer motif (PAM) essential for binding to Cas9 (Naito et al. 2015). The PAM sequence is located 5’ to the target DNA sequence and is necessary for Cas9to bind to the target DNA (Berraaouan 2017). The guide RNA also contains a sequence that is coresponding to the direct  DNA. This sequence is used to identify and bind to the target DNA sequence. Once the gRNA binds to the target DNA, it directs Cas9 to cleave the target DNA (Manghwar et al. 2020). The Cas9 nuclease domain contains two nuclease domains: the HNH domain and the RuvC domain (Ma, Zhang and Huang 2014). The HNH domain cleaves the phosphate backbone of the target DNA, whereas the RuvC domain cleaves the sugar-phosphate backbone (Huang 2020). This results in a double-strand DNA break (DSB) which is then restored by various mechanisms, including non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Devkota 2018).

The CRISPR-Cas9 system

The CRISPR-Cas9 system is a versatile and efficient genome engineering tool used for different functions, like gene knockouts, gene insertions, and epigenetic modifications (Ding et al. 2016). The CRISPR-Cas9 system comprises two main components: the Cas9 protein and the guide RNA (Ling et al. 2020). The Cas9 protein is a nuclease that cleaves DNA at specific target sequences, and the guide RNA is a short RNA molecule that directs the Cas9 protein to the correct target sequence (Peng, Lin and Li 2016). The specificity of the CRISPR-Cas9 system is due to the guide RNA. The guide RNA contains a sequence that is complementary to the target sequence, which binds to the Cas9 protein (Briner et al. 2014). When the guide RNA and the target sequence bind to each other, the Cas9 protein is activated and cleaves the DNA at the target site.

A range of creatures, including bacteria, plants, and animals, have had their genomes edited using the CRISPR-Cas9 system. The CRISPR-Cas9 system has been used to delete genes, insert genes, and modify gene expression. The CRISPR-Cas9 system is a simple and efficient way to modify an organism’s genome, and it has the potential to be used for many different applications. It is important to note that the CRISPR-Cas9 system can cause off-target effects, and it is important to consider these effects when using the CRISPR-Cas9 system (Wolt et al. 2016).

The guide RNA is a sequence of RNA specifically designed to match the target DNA sequence (Naito et al. 2015). The guide RNA contains a sequence of base pairs complementary to the target DNA sequence (Nishimasu et al. 2014). When the CRISPR-Cas9 system is used to edit the genome, the guide RNA binds to the target DNA sequence and directs the Cas9 enzyme to the correct location. This ensures that the Cas9 enzyme will only cleave the target DNA sequence and not any other DNA sequences in the genome. It is important to note that the guide RNA does not need to be perfect, as the Cas9 enzyme can still cut DNA strands if there are mismatches in the guide RNA sequence (Lin et al. 2014).

When the CRISPR-Cas9 system is used to edit the genome, the Cas9 enzyme is guided to the target DNA sequence by the guide RNA. Once it reaches the target sequence, the Cas9 enzyme cleaves the DNA strands, causing a double-strand DNA break. The double-strand DNA break can cause a variety of cellular responses, including DNA repair, cell death, or genetic alterations (Meyn 1995). The type of cellular response that occurs depends on the organism and the location of the target sequence in the genome.

Eukaryotic cells, including animals and plants, have a more complex genome than prokaryotic cells, including bacteria (Bestor 1990). Eukaryotic cells have several mechanisms that protect the genome from being edited by the CRISPR-Cas9 system. One such mechanism is the use of endogenous RNA interference (RNAi), which is a process that helps to silence genes. When the CRISPR-Cas9 system is used to edit the genome, the cells can activate endogenous RNAi to silence the edited genes (Kim and Rossi 2008). This can have unfavorable implications, such as cell death or genetic changes.

It is vital to remember that not all cells react the same way to the CRISPR-Cas9 system. Some cells may repair the double-strand DNA break, while others may undergo genetic alterations or die. The type of response that a cell undergoes depends on the organism and the location of the target sequence in the genome (Bestor 1990).

Deleting genesis one of the primary function of the CRISPR-Cas9 system. Researchers have used the CRISPR-Cas9 system to delete genes of different organisms, including bacteria, plants, and animals. The CRISPR-Cas9 system is used to implant genes into the genomes of bacteria and plants. Researchers have recently started using the CRISPR-Cas9 system to modify animal gene expression.

The CRISPR-Cas9 system has several advantages over other genome editing methods, such as zinc fingers and TALENs (Jang et al. 2018). The CRISPR-Cas9 system is easy to use and is relatively inexpensive. The CRISPR-Cas9 system edits the genomes of a variety of organisms, including bacteria, plants, and animals (Xing et al. 2014). CRISPR-Cas9 system can cause unintended mutations in the genome, thus thorough testing should be done before using the system to edit the human or other organisms genomes (Manghwar et al. 2020).

The CRISPR-Cas9 system is a strong gene-editing tool (Manghwar et al. 2020). The guide RNA directs the Cas9 enzyme to the specific DNA target. The Cas9 enzyme then cleaves the DNA target, resulting in a double-strand DNA break. The CRISPR-Cas9 system has revolutionized genome engineering (Filippova et al. 2019). The CRISPR-Cas9 system is a simple and efficient way to modify the genomes of organisms.

Different methods of delivering CRISPR-Cas9 into cells

A CRISPR-Cas9 system is a versatile tool for genome engineering that can modify the genomes of a variety of organisms, including bacteria, plants, and animals (Zhu, Holmes, Aronin and Brodsky 2014). The specificity of the CRISPR-Cas9 system is due to the guide RNA, which directs the Cas9 enzyme to the desired DNA target. There are several different methods for delivering the CRISPR-Cas9 system into cells, including using vectors such as viruses or plasmids, electroporating cells with CRISPR-Cas9 complexes, or injecting them directly into cells (Prykhozhij et al. 2015). For instance, the CRISPR-Cas9 system has been used to engineer human T cells to resist HIV infection. The CRISPR-Cas9 system was also used to correct a genetic defect in human embryos (Song 2017). This study showed that the CRISPR-Cas9 system could be used to correct a gene mutation that causes beta-thalassemia, a blood disorder. However, the CRISPR-Cas9 system has also been associated with unintended consequences, such as off-target effects and insertional mutagenesis.

Off-target effects occur when the Cas9 enzyme cleaves DNA targets other than the desired target. This can result in unwanted mutations in the genome when using the CRISPR-Cas9 system for gene editing (Jacinto and Ferreira 2020). One way to reduce off-target effects is to use Cas9 variants that have been modified to be more specific.

Insertional mutagenesis occurs when the Cas9 enzyme inserts the genome at a random location (Manghwar et al. 2020). This can result in the insertion of new genes or mutations in existing genes. Insertional mutagenesis is also a major concern when using the CRISPR-Cas9 system for gene editing. One way to reduce insertional mutagenesis is to use Cas9 variants that have been modified to be less active (Manghwar et al. 2020). Despite these reservations, the CRISPR-Cas9 system is a strong genome editing tool that has been used to change the genomes of a variety of animals (Manghwar et al. 2020). The CRISPR-Cas9 system will likely continue to be used for genome engineering in the future.

CRISPR-Cas9 development and applications for genome engineering

CRISPR-Cas9 is a versatile and effective genome engineering tool widely used for various purposes such as gene knockouts, gene insertions, and epigenetic modifications (Filippova et al. 2019). The CRISPR-Cas9 system comprises two main components: the Cas9 protein and the guide RNA are two components of the Cas9 system. The Cas9 protein is in charge of identifying and cleaving the DNA target, while the guide RNA points Cas9 in the right direction.

One of the major advantages of the CRISPR-Cas9 system is its high specificity. The specificity of the CRISPR-Cas9 system is due to the guide RNA. The guide RNA recognizes the target DNA sequence and directs Cas9 to the site of interest (Naito et al. 2015). This ensures that the Cas9 protein will only cleave the desired DNA target. In addition, the CRISPR-Cas9 system can be easily modified to increase its specificity. The CRISPR-Cas9 system has been successfully used to delete genes, insert genes, and modify gene expression. For example, the CRISPR-Cas9 system was used to edit the genome of a human embryo (Song 2017). The CRISPR-Cas9 system was also used to correct a genetic mutation in a patient with sickle cell anemia.

The CRISPR-Cas9 system has also been used for basic research purposes. For example, the CRISPR-Cas9 system was used to study the function of specific genes in bacteria. The CRISPR-Cas9 system was also used to study the effects of gene knockouts on animal development (Jang et al. 2018). Overall, the CRISPR-Cas9 system is a reliable and adaptable tool that has been employed for gene knockouts research, gene insertions, and genetic mutations, among other things (Jang et al. 2018).

The CRISPR-Cas9 system’s cellular reaction

When the CRISPR-Cas9 system cleaves DNA, it can cause two types of DNA damage: double-stranded breaks (DSBs) and single-stranded gaps. The cell’s reaction to different types of DNA damage is influenced by several parameters, including the location of the break, the cell type, and the cell cycle stage (HDR) (Devkota 2018). DSBs are more harmful to the cell than single-stranded gaps, and the cell’s response to DSBs can vary depending on the type of cell. In some cells, such as bacteria, DSBs are repaired by a process called homology-directed repair (HDR) (Devkota 2018). In other cells, such as mammalian cells, DSBs are repaired by a process called non-homologous end joining (NHEJ) (Schiermeyer et al. 2019). NHEJ is a less accurate repair process and can lead to mutations (Devkota 2018).

Cas9 cleaves DNA at specific locations that are determined by the guide RNA. The guide RNA contains the sequence of the DNA target that Cas9 will recognize and cleave. Therefore, the specificity of the CRISPR-Cas9 system is due to the guide RNA. There are many ways to modify the guide RNA sequence to change the specificity of the CRISPR-Cas9 system. For example, scientists can use a computer program to design guide RNAs that will cleave specific DNA targets. They can also modify the guide RNA sequence to make it more or less specific.

Conclusion

The CRISPR-Cas9 system is a simple and efficient way to modify an organism’s genome, and it has the potential to be used for many different applications (Zhu, Holmes, Aronin and Brodsky 2014). It is important to note that the CRISPR-Cas9 system can cause off-target effects, and it is important to consider these effects when using the CRISPR-Cas9 system. The cell’s response to the CRISPR-Cas9 system depends on some factors, including the location of the break, the type of cell, and the stage of the cell cycle (HDR) (Devkota 2018). DSBs are more harmful to the cell than single-stranded gaps, and the cell’s response to DSBs can vary depending on the type of cell. In some cells, such as bacteria, DSBs are restored by a procedure termed homology-directed repair (HDR) (Schiermeyer et al. 2019). In other cells, such as mammalian cells, DSBs are repaired by a process called non-homologous end joining (NHEJ). NHEJ is a less accurate repair process and can lead to mutations (Manghwar et al. 2020). Scientists can use a computer program to design guide RNAs that will cleave specific DNA targets or modify the guide RNA sequence to make it more or less specific. The CRISPR-Cas9 system can be utilized in many different areas, and it is important to take into account the potential off-target effects when using the CRISPR-Cas9 system.

References

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Bestor, T.H., 1990. DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes. Philosophical Transactions of the Royal Society of London. B, Biological Sciences, 326(1235), pp.179-187.

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Devkota, S., 2018. The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis. BMB reports, 51(9), p.437.

Ding, Y., Li, H., Chen, L.L. and Xie, K., 2016. Recent advances in genome editing using CRISPR/Cas9. Frontiers in plant science, 7, p.703.

Filippova, J., Matveeva, A., Zhuravlev, E. and Stepanov, G., 2019. Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems. Biochimie, 167, pp.49-60.

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Huang, X., Sun, W., Cheng, Z., Chen, M., Li, X., Wang, J., Sheng, G., Gong, W. and Wang, Y., 2020. Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2. Nature communications, 11(1), pp.1-14.

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