Mediated Genomic Editing Techniques Used By Bacteria And Archea As An Adaptive Defense Mechanism

CRISPR Cas9 has many advantages, but it has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods. CRISPR reduces the time required to modify targeted genes and has improved bioinformatics tools to identify the best sequences to design guide RNAs. CRISPR Cas9 in embryos works extremely well for the generation

Phages are the most common organisms on Earth (3,7): they infect bacteria by injecting single-stranded DNA into their host cell, thereby altering the genetic code of the prokaryote. Once transcribed and translated, the injected DNA changes the function of the cell, often killing the host cell in the process (3). Over eons, archaea and some bacteria have adapted to such attacks with CRISPR-Cas systems, also known as clustered regularly-interspaced short palindromic repeats. The bacteria incorporate invading viral DNA into their genetic material in between redundant palindromic sequences. The viral DNA is then transcribed into pre-crRNA, where it combines with a Cascade complex. Cascade proteins come from CRISPR-associated (Cas) genes. Such genes code for small enzymes responsible for cleaving. They often join together into larger complexes known as Cascade, or CRISPR-associated complex for antiviral defense (2). The cas proteins use the RNA template as a guide and cut any other genetic material that matches that of the viral-template RNA, preventing the corrupt viral DNA from being transcribed into mRNA. As the number of spacers in the bacterium’s DNA increases, the level of phage resistance increases as well (1). Insofar, understanding of CRISPR and Cas genes is very limited. It is very much a twenty-first century discovery.

CRISPR immunity involves the three following phases: acquisition, expression and interference. During the acquisition stage, new spacer sequences are incorporated into the CRISPR locus. During the expression stage, the CRISPR array is transcribed into a premature crRNA (pre-crRNA) and then matures into the CRISPR RNA (crRNA). In the last stage, the foreign DNA is destroyed using the processed crRNA in some form of effector complex containing Cas proteins (Weidenheft et al., 2012) (Figure 3).

CRISPR-Cas9 is making many advances in the medical and science field with changing genes in babies and plants. By doing this, doctors and scientists are able to get rid of diseases and human viruses in animal zygotes, human cells, and human embryos. CRISPR-Cas9 is making it easier to get rid of certain genetic disorders and deadly human diseases in babies. In doing this, we can lessen the chance of the spread of certain diseases. Which in turn can keep the world population on the ultimate rise.

The endonuclease Cas9 is the core multifunctional protein of the CRISPR-Cas9 system. Two nuclease domains: HNH (39) and RuvC-like (40), within the Streptococcus pyogenes Cas9 (SpCas9) protein conduct the function of cleaving DNA strands. Studies have confirmed that the tracrRNA: crRNA duplex structure will lead to the sequence-specific DNA cleavage (41). PAM short sequence facilitates crRNA to recognize the targeted DNA sequence (42). Cas9 protein cuts the specific DNA strand that is complementary to the 20bp crRNA sequence via HNH domain; on the other hand, the SpCas9 RuvC-like domain cleaves the opposite targeted DNA strand (42) (Fig. 4). Mutating either of these two nucleases domains of SpCas9 will introduce a single-stranded DNA nick, while mutating both HNH and RuvC-like domains will transform SpCas9 into a RNA-guided DNA binding protein (42). X-ray crystallography and has confirm that endonuclease Cas9 undergoes a series of conformational rearrangements when it binds to the guide RNA and target DNA (55).

Cas 9 cleaves the target DNA and creates a Double Strand Break (DSB) that can then be repaired by DNA repair pathway. The primary DNA repair pathway in eukaryotic cells is Non-Homologous End Joining (NHEJ). NHEJ ligates the broken DNA strands together which results in insertions or deletions which lead to gene inactivation. Another DNA repair pathway is Homology Directed Repair (HDR), which is a precise process, usually restricted to dividing cells, that uses exogenous DNA template and allows for desired sequence to be added to the gene (Reis 2014; Wang 2016). The most common result of CRISPR/Cas9 were deletions (Koike 2013). It was observed that HDR was only achieved with simultaneous introduction of repair donor DNA, Cas9, and guide RNAs (Mali Yang 2013). Researchers have altered the domains of Cas9 to get different forms with differing functions. The mutant form Cas9D10A only has nickase activity. This increases the precision of Cas9 by making two Cas9 make a single stranded break to create the double stranded break. This allows for less off target damage because the likely hood of two Cas9 both reacting in off target locations together is rare. This also helps encourage NDR instead of NHEJ. A third Cas9, dCas9, has been developed by mutating the domains of Cas9 to deactivate the DNA cleavage but allow it to remain bound to DNA. This allows blockage of transcription by sterical hindrance, marking DNA in the cell, and adding transcription factors to alter expression (Reis

CRISPR-Cas9, often called “CRISPR” is a genome-editing tool used in the field of genome engineering. CRISPR stands for Clustered Regularly Interspaced Short Palindromic repeats, and combined with Cas9, the two are an important part of a bacteria defense system. The goal of this technique is to target specific portions of genetic code and to edit the DNA at exact locations. CRISPR is natural to the bacterial cell’s immune system and provides protection against viruses. This allows researchers and scientists to modify genes in living cells and organisms (“Questions and Answers about CRISPR”, 2017).

Additionally, CRISPR-Cas mediated immunity develops through the intake of DNA from genetic elements such as plasmids and phages followed by its incorporation into the CRISPR loci. It is within the CRISPR loci where the small interfering RNAs are processed and transcribed that aid the nucleases for precise cleavage of corresponding sequences. Interestingly enough, recognizing the genetic sequences in CRISPR loci give a clear insight into vaccinations events that the prokaryote would’ve gone through, and can give some perception in the environmental changes occurring over a period of time. Therefore, cas endonucleases, which can be reengineered by small guide RNAs, have proven to demonstrate remarkable development and flexibility for genome editing and can be repurposed for a number of DNA purposing applications. The main scope of the report will focus on the structure and function of the CRISPR-CAS system, the organisation of the CRISPR loci and its significance, as well as contrasting some differences between prokaryotes in CRISPR guide RNA (crRNA) design.

The RNA-guided CRISPR-Cas9 system has revolutionized genetic engineering by allowing targeted genomic modification through the simple design of a 20 base pair guiding sequences. This groundbreaking technology utilizes a short guide RNA (sgRNA) to direct the Cas9 nuclease to a specific genomic locus through complementary base pairing. The guide RNA or sgRNA is responsible for the specificity of the CRISPR-Cas9 system, and several consideration need to be taken during its design process. In bacteria and archaea, the crRNA and tracrRNA form a complex which acts as the homing device for directing the Cas9 nuclease to the invading foreign genetic materials. The tarcrRNA 's scaffolding ability along with the specificity of the crRNA can be

Clustered regularly interspaced short palindromic repeats (CRISPR) is a microbial adaptive immune system which utilizes an RNA guided Cas9 endonuclease to specifically target and destroy viral DNA via double stranded breaks (Cong et al., 2013; Hsu et al., 2014). Researchers discovered that they could manipulate the guide RNA (gRNA) to deliver Cas9 to a DNA sequence of interest within the human genome. This enables Cas9 to induce site specific double stranded breaks (Hsu et al., 2014). Donor DNA sequences containing fully functional genes ending in DNA that is homologous to the cleaved ends can then be incorporated into the break via homologous directed repair (HDR) by the cell’s own DNA-repair mechanism (Mali et al., 2013). In this way,

CRISPR loci are first identified in archaea and bacteria when they systematically drew attention from scientists with their biological function to fight phages and viruses (Hsu, Lander, Zhang, 2014). Structurally, a clustered set of Cas (CRISPR-associated) genes and a unique CRISPR array constitute the CRISPR loci. The CRISPR array was further comprised of short repetitive sequence interspaced by distinctive sequences (spacers) in correspondence with exogenous genetic bits (protospacer). The natural CRISPR systems in bacteria and archaea carried out their adaptive antiviral immunity by following a three-step mechanism, namely acquisition of spacers, crRNA biogenesis, and interference (Wright, Nuñez, Doudna, 2016).

CRISPR-CAS works by transcribing a CAS9 nuclease protein (from CAS genes) and a guide RNA (crRNA) from spacers within the CRISPR loci. Spacers are sections of copied “foreign” DNA that have been inserted from previous exposures to phages or antibiotic gene transfers with other bacteria. When a foreign piece of

The story of this novel technique began in 1987 when Japanese researchers Nakata and his colleagues observed a series of 29nt DNA repeats interspaced by five 34nt sequences downstream of the iap enzyme gene in Escherichia coli (19). More and more such repetitive sequences were detected in bacteria and archaea strains over time. The constant findings have sparked research interest in these repetitive elements. In 2002, Mojica and Jansen abbreviated these clustered regularly interspaced short palindromic repeats as CRISPR (20). In the same year, Jansen also reported that three distinct types of CRISPR-associated Cas genes were usually adjacent to repetitive DNA sequences (20); later, those three Cas genes were utilized to classify the CRISPR system (CRISPR type I-III). The major difference of three CRISPR system is that multiple complex Cas protein components constitute type I and III; in contrast, type II is only constructed by one single protein: Cas9 (Fig. 3) (21). Even though increasingly molecular structures of CRIPSR systems had been understood, their underlying biological mechanisms and functions were still a

CRISPR systems are generally delivered in the cell using normal methods of transfection such as microinjection, gene gun, electroporation, sonoporation, and using viruses such as adenoviruses and lentiviruses. In cultured mammalian cells, researchers have used electroporation, nucleofection, and Lipofectamine mediated transfection methods to deliver vectors expressing the gene for gRNAs and Cas9 endonuclease. In cultured human and mouse cells, Lentiviral vectors have also been used as a mode of delivery of genes. In addition to animal models and cell lines, Cas9 has been used successfully in a lot of plant species including wheat, rice, sorghum, tobacco, and thalecress using a range of standard delivery methods such as PEG-mediated transformation of protoplasts, Agrobacterium-mediated transfer in embryos and leaf tissue, and/or bombardment of callus cells with plasmid DNA. [1]

Fig. 1. Demonstration of Type II CRISPR-Cas systems with Cas9 protein: (1) When a new virus infects a bacteria, its genome which will convert from RNA to double stranded DNA into the bacteria. Using the genetic information, new DNA spacers will be created. (2) Cas genes produces Cas protein through transcription and translation. Meanwhile, the new spacer sequence will be transcribed into CRISPR RNA (crRNA). (3)Trans-activating crRNA (tracrRNA) which acts as a guidance in searching target DNA is also formed and combine with Cas9 protein. (4) crRNA and tracrRNA which bind with Cas9 protein are hybridized. (5) The complex will then search for, bind with and unwind the target double stranded DNA sequence. (6) Finally, the target DNA sequence will be sliced up by the Cas9 complex (Sakuma & Yamamoto, 2015).