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BREIF ON MOLECULAR SCISSORS
ABSTRACT
Advances in genome have revolutionized modern biology, enabling precise control over genetic information in living organism. Molecular Scissors are gene-editing tools used in genetic engineering to modify DNA sequences through deletion, insertion, knockout, or point mutation. Techniques like CRISPR-Cas, TALENs, and Zinc finger nucleases (ZFNs) target specific genomic regions by inducing double strand DNA breaks, enabling precise gene modifications through the cell’s natural repair mechanisms. The specific repair pathway activated, play a crucial role in shaping the final editing outcome. These are resolved by the cell, through endogenous pathways, such as non-homologous end-joining (NHEJ), and homologous recombination (HR), micro-homology-mediated end-joining (MMEJ). In certain contexts, base-excision repair (BER), and mismatch repairs (MMR) may also assist in correcting. Sequence level changes. These gene techniques have significantly advanced our ability to study genetic diseases and have transformative applications in biotechnology, agriculture, and medicine (Doudna & Charpentier, 2014; Komor et al., 2016).
INTRODUCTION
The term molecular scissors refer to enzymes or engineered tools capable of cleaving DNA or RNA at specific locations. These tools are at the forefront of biotechnology and genetic engineering, enabling precise manipulation of genetic material. Among them, CRISPR-Cas9 has emerged as a revolutionary system that allows researchers to edit genes with high efficiency and accuracy (Jinek et al., 2012). Originally part of the bacterial immune system, CRISPR- Cas9 was adapted to serve as a versatile genetic editing platform in eukaryotic cells. The CRISPR-Cas9 system comprises two key components: the Cas endonuclease, which acts as the molecular “scissors,” and a guide RNA (gRNA), which directs Cas9 to a specific DNA sequence for cleavage. This programmable approach has made CRISPR-Cas widely accessible and effective in a variety of biological applications, from basic research to therapeutic development (Doudna & Charpentier, 2014). In addition to its biomedical relevance, CRISPR is also transforming agriculture by enabling the creation of crops with improved yield, disease resistance, and environmental adaptability. By offering a cost-effective and scalable gene-editing solution, it is accelerating discoveries in genetic research and promoting innovation across scientific disciplines.
While restriction enzymes are classified as endonucleases, CRISPR- Cas systems originate from bacterial immune mechanisms and are not categorized as restriction enzymes, even though they function similarly by introducing site-specific double-stranded breaks. Molecular scissors can be divided into two types based on cutting locations -Example: exonuclease I and lambda exonuclease.
MOLECULE SCISSORS
ENDONUCLEASE
-Example: CRISPR Cas, Restriction endonuclease and TALENs
EXONUCLEASE
ENDONUCLEASE
Endonucleases are enzymes that cleave phosphodiester bonds within the internal regions of DNA or RNA molecules. They are known for their high efficiency and precision in cutting at specific target sequences. These enzymes are central to many genetic engineering techniques because they allow for precise DNA modification. Key Examples: CRISPR-Cas Restriction endonucleases such as EcoRI and HindIII TALENs Mechanisms: EcoRI recognizes the palindromic DNA sequence GAATTC and cuts between G and A, generating sticky ends. HindIII targets the sequence AAGCTT and cuts between A and T, also producing sticky ends. Each type of endonuclease has its unique recognition site and cutting mechanism, making them suitable for different molecular biology applications. Exonuclease Exonucleases remove nucleotides one at a time from either the 3’ or 5’ end of DNA or RNA strands. Unlike endonucleases, they do not cut internally but instead digest nucleic acids from the ends. These enzymes are essential in processes like DNA replication, repair, and proofreading. Key Examples: Exonuclease I – cleaves single-stranded DNA from the 3’ end in a 3’ to 5’ direction Lambda exonuclease – removes nucleotides from double- stranded DNA starting at the 5’ end
Fig 2. CRISPR Class 1 Class 1 is more widely present in about 90% of the CRISPR Cas loci, uses crRNA for guidance for specific locations TYPE I Cas 3 is an enzyme that is situated with the immune system of bacteria; it forms a multi-protein complex called ‘CASCADE’ which it uses to cleave foreign DNA. It performs various functions like DNA degradation, acting as a nuclease. It can also offer long-strand deletion where it can cut or chew long strands in one direction from a specific point. TYPE III – Cas 10 acts as a ribonuclease that is specially designed to cleave RNA molecules. Cas 10 has a variety of functions such as transcribing CRISPR array spacer repeat sequences into long precursors (pre- crRNA). crRNA guides the cas10 complex to the foreign DNA or RNA sequences which is trying to enter into the cell or harm the cell in any possible way and the complex with other proteins like Csm or Cmr degrades or disintegrates the sequences after binding to them which gives it a special ability to target phage. It also helps the bacteria as an antiviral defense system, it also helps with gene regulation where it can be used to regulate the gene expressions which makes it potential for therapeutic research applications, it can
TYPE I TYPE IV TYPE II TYPE^ IV
TYPE III TYPE^ V
also be used in the development of biosensors that can help in detecting specific nucleic acids. Cas 10 also shows the future possibilities in helping with the development of “Antimicrobial therapies” and phage engineering. TYPE IV – Cas 7 is a single subunit DNA nuclease. Like Cas 10, Cas 7 also provides a defense or immune mechanism to bacteria against the action of bacteriophage by probably degrading the foreign gene elements with the guidance of crRNA with its protein complex it attaches to the inhibitor strands and strands breaking and degrading it making it from transmission to stop which can harm the bacteria, it also helps with DNA repair within the bacterial cell. As it's not that used like Cas 9 so information related it Cas 7 is limited but from the research studies Cas 7 shows future potential in DNA manipulation.
Cas 12 (1300 amino acids) has a smaller size in comparison to Cas which makes it easier to package and deliver into cells and due to the presence of more PAM (protospacer adjacent motif- is a short specific sequence which follows the Cas and is present near the target site of DNA sequences which needs to be cut) sequences it is more flexible and easier for it to target specific DNA locations and has been categorized to have the ability to degrade non-specific ssDNA (single-strand DNA) strands into single or double-strand nucleotides of all sorts available without any discrimination and forms staggered ends to prevent degradation of unrelated or unnecessary stand a mechanism is used where the guidance crRNA is used as databases search it found to give the solution of using crRNA in sufficient concentration with the help of crRNA molecules forming a new interference complex from the cleaved R loop it can revert the active conformation resulting in stopping or shut down of unnecessary or unspecific degradation or activity. Cas 12a has also successfully made corrections in mammalian cells which causes muscular dystrophy due to mutation in patient of induced pluripotent stem cells. TYPE VI – Cas 13 is an RNA-targeting nuclease, with its recent discovery scientists have found it to make precise manipulation or change in RNA without having any permanent changes in the genome which can make it a good tool for research purposes to study and research. Cas 13 has also been used and seen as successful in the production of virus-resistant plants. Cas 1 also uses crRNA as its guide protein which helps it to bind to “single-strand RNA”. RNA interference, RNA detection, RNA editing, and RNA targeting are some Cas 13 RNA technology-based applications that are used differently depending
on the various organisms. Cas 13 has also been used as a biosensor with its unique properties it has been successfully used to detect viruses, bacteria, and even cancer tumor cells and help in the early detection of the tumor cells a new technology has been developed that can also use to detect miRNA (group of small non-coding RNAs) which is contained by EV (extracellular vesicles) which are originated from intercellular transport system which helps in signaling and organelle function interaction for them. Cas13 also shows future possibilities to be helpful and used in therapeutics and biotechnology and research.
RESTRICTION ENZYMES
Restriction enzymes or Restriction endonucleases were discovered in the early 1960’s and are proteins that act as genetic/molecular scissors that alter or make cuts on a specific site of the genome using site-specific protein-based scissors that cleave the DNA nucleotides is produced by bacteria. Restriction enzymes have been used to manipulate or alter the particular DNA sequence of the organism even though not too precise cuts are made in comparison to CRISPR. being an endonuclease, restriction enzymes also target the sites anywhere in the middle of the DNA sequence between 3’ to 5’ site. Restriction enzymes also used to be part of bacterial defense mechanism where they used to target foreign bodies like bacteriophages by cutting the phage DNA into pieces which entered the bacterial and eliminating it to prevent it to over take the bacterial body and prevent it from eventually killing bacteria. Every restriction enzyme have a particular restriction site, restriction sites are the sites where the particular specific enzymes recognize and binds to do the processes. There are four types of restriction enzymes- Type 1- they were the first restriction enzymes to be discovered and purified, these have separate (R), (M) and (S) subunits. They are said to have a unique ability of shifting the domain or recognition site which the protein comes and binds eg. EcoKI, EcoBI. Type 2-These restriction enzymes are the most widely used due to their high precision and efficiency rate have been found to be compatible with being used to do DNA fingerprinting, DNA cloning, and many more applications. eg BamHI, EcoRI Type 3- they have a unique ability to recognize even non-
palindromic sequences but are not that widely known or used in comparison to Type 1 and Type 2 restriction enzymes, usually used to study the bacterial defense mechanism. eg. EcoP1I, EcoP15I Type 4- eg. Mcr, Mrr these are modification dependent and is continuously undergoing evolution but is not that widely known or worked on.
TALENS
TALENs is non-specific targeting molecular scissors which by doing the alteration and modification with binding with a DNA recognizing domain have attained the ability to recognize and target sites specified for the job. It is an artificially engineered type of restriction endonuclease, being an endonuclease, it also targets the sites in between the 5’ and 3’ ends and cleaves through between them chopping or chewing off the sequences specified in the DNA and bind to any nucleotide, can bind to 36 base pair sequence. It was widely used genetic scissors before the discovery or use of CRISPR. The cuts made by the TALENs can be repaired by the cell’s own mechanism, TALENs is tend to be more flexible in applications than CRISPR and can be used in diverse cell types. it has the ability to work on almost any DNA sequence possible. Depending on the usability it can be engineered or altered depending on the target site. It also has some limitations like it an bind to any unnecessary DNA site not required for the processes and designing and engineering it can be hard for some specific sites compared to other genome editing techniques.
precision and efficiency and it tends to be less flexible while being used in these types of situations and its modification is also considered not easy compared to the other methods which can be used to perform on the same action It can be used to study research, biotechnology and genetic engineering and to replicate, eliminate, and modify DNA sequences. Meganucleases can be found in a quite a large amount of organisms like archaebacteria, fungi, algae and even some plants. It mainly has two main enzymes; intein endonuclease and intron endonucleases first identified in 1990’s making a few minor changes to the amino acid sequence and then selecting the functional proteins based on changes to the natural recognition site to alter the specificity of current available meganucleases. An even more extreme approach has been to take use of a feature that contributes significantly to the naturally high degree of variety of meganucleases: the ability to associate or fuse protein domains from distinct enzymes, having this method, chimeric meganucleases having a novel recognition site made up of half-sites from protein B and meganuclease A can be created. Using this technique, two chimeric meganucleases, DmoCre and E-Drel, were produced by combining the protein domains of I-DmoI and I-CreI. Although methods based on site-specific recombination are being developed, other teams have been trying to increase the efficiency of homologous gene targeting. Clearly, two main areas of research are being identified: (i) methods we will call "matrix optimisation," which essentially entail modifying the targeting vector structure to optimise efficacy, and (ii) methods that utilise additional effectors, usually sequence-specific endonucleases, to facilitate homologous recombination (HR). In the field of matrix optimisation, several
different approaches have been tried, with varying degrees of success. For example, attempts to replace the DNA repair matrices used in classical gene targeting experiments with chimeric DNA- RNA modified oligonucleotides, including different modifications and/or specific secondary structures, were initially thought to be a promising approach, but ultimately proved to be ineffective in numerous.
positioned on the top and bottom strands of the DNA substrate in a head-to-tail orientation. CONCLUSION Molecular scissors is a technique that helped genetics genetic engineering and biotechnology give a lot of new research we observed the old and the new used tools or restriction enzymes we noticed how much more we need to work on to get the results the future is asking for. Genome sequencing undoubtedly helps us understand the genetic code responsible for inherited illnesses. However, the cure for genetic diseases requires permanently rectifying the mutation in the gene that causes the illness, rather than merely giving patients drugs to temporarily alleviate the harmful effects of the condition. Be it CRISPR or other restriction enzymes a lot of information and research needs to be done on the new developing or already developed techniques it also shows how much of an opportunity genetic engineering holds with further research there are future possibilities to even solve global issues of Cancer but for that lot needs to be done but it's still fascinating t have the idea of what not these new developing molecular scissors can probably a human clone of therapeutics for many diseases which might seem like impossible to treat now might not be so hard in the coming future and in the world of development
