Insert (molecular biology)

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Inserted sequence Insertion sequence.jpg
Inserted sequence

In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism. [1]

Contents

Inserts can range from physical nucleotide additions using a technique system or the addition of artificial structures on a molecule via mutagenic chemicals, such as ethidium bromide or crystals.

Inserts into the genome of an organism normally occur due to natural causes. These causes include environmental conditions and intracellular processes. Environmental inserts range from exposure to radioactive radiation such as Ultraviolet, mutagenic chemicals, or DNA viruses. Intracellular inserts can occur through heritable changes in parent cells or errors in DNA replication or DNA repair.

Gene insertion techniques can be used for characteristic mutations in an organism for a desired phenotypic gene expression. A gene insert change can be expressed in a large variety of ends. These variants can range from the loss, or gain, of protein function to changes in physical structure i.e., hair, or eye, color. The goal of changes in expression are focused on a gain of function in proteins for regulation [2] or to termination of cellular function for prevention of disease. [3] The results of the variations are dependent on the place in the genome the addition, or mutation is located. The aim is to learn, understand, and possibly predict the expression of genetic material in organisms using physical and chemical analysis. To see the results of genetic mutations, or inserts, techniques such as DNA sequencing, gel electrophoresis, immunoassay, or microscopy  can observe mutation.

History

The field has expanded significantly since the publication in 1973 with biochemists Stanley N. Cohen and Herbert W. Boyer by using E. coli bacteria to learn how to cut fragments, rejoin different fragments, and insert the new genes. [4] The field has expanded tremendously in terms of precision and accuracy since then. Computers and technology have made it technologically easier to achieve narrowing of error and expand understanding in this field. Computers having a high capacity for data and calculations which made processing the large volume of information tangible, i.e., the use of ChIP and gene sequence.

Techniques and protocols

Homology directed repair (HDR) is a technique repairs breaks or lesions in DNA molecules. The most common technique to add inserts to desired sequences is the use of homologous recombination. [5] This technique has a specific requirement where the insert can only be added after it has been introduced to the nucleus of the cell, which can be added to the genome mostly during the G2 and S phases in the cell cycle. [6]

CRISPR gene editing

CRISPR gene editing based on Clustered regularly interspaced short palindromic repeats (CRISPR) -Cas9 is an enzyme that uses the gene sequences [7] to help control, cleave, and separate specific DNA sequences that are complementary to a CRISPR sequence. [8] [9] These sequences and enzymes were originally derived from bacteriophages. [10] The importance of this technique in the field of genetic engineering is that it gives the ability to have highly precise targeted gene editing and the cost factor for this technique is low compared to other tools. [11] [12] [13] The ability to insert DNA sequences into the organism is easy and fast, although it can run into expression issues in higher complex organisms. [14] [15]

Transcription activator-like effector nuclease

Transcription activator-like effector nuclease, TALENs, are a set of restriction enzymes that be created to cut out desired DNA sequences. [16] These enzymes are mostly used in combination with CRISPR-CAS9, Zinc finger nuclease, or HDR. The main reason for this is the ability for these enzymes to have the precision to cut and separate the desired sequence within a gene.

Zinc finger nuclease

Zinc finger nucleases are genetically engineered enzymes that combine fusing a zinc finger DNA-binding domain on a DNA-cleavage domain. These are also combined with CRISPR-CAS9 or TALENs to gain a sequence-specific addition, or deletion, within the genome of more complex cells and organisms. [17]

Gene gun

The gene gun, also known as a biolistic particle delivery system, is used to deliver transgenes, proteins, or RNA into the cell. It uses a micro-projectile delivery system that shoots coated particles of a typical heavy metal that has DNA of interest into cells using high speed. The genetic material will penetrate the cell and deliver the contents over a space area. The use of micro-projectile delivery systems is a technique known as biolistic. [18]

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

<span class="mw-page-title-main">Designer baby</span> Genetically modified human embryo

A designer baby is a baby whose genetic makeup has been selected or altered, often to not include a particular gene or to remove genes associated with disease. This process usually involves analysing a wide range of human embryos to identify genes associated with particular diseases and characteristics, and selecting embryos that have the desired genetic makeup; a process known as preimplantation genetic diagnosis. Screening for single genes is commonly practiced, and polygenic screening is offered by a few companies. Other potential methods by which a baby's genetic information can be altered involve directly editing the genome before birth, which is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins Lulu and Nana were edited as embryos, causing widespread criticism.

<span class="mw-page-title-main">CRISPR</span> Family of DNA sequence found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

Gene editing may refer to:

A guide RNA (gRNA) is a piece of RNA that functions as a guide for RNA- or DNA-targeting enzymes, with which it forms complexes. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or DNA. They occur naturally, serving important functions, but can also be designed to be used for targeted editing, such as with CRISPR-Cas9 and CRISPR-Cas12.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.

<i>Fok</i>I

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.

<span class="mw-page-title-main">Transcription activator-like effector nuclease</span>

Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

Prime editing is a ‘search-and-replace’ genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

GUIDE-Seq is a molecular biology technique that allows for the unbiased in vitro detection of off-target genome editing events in DNA caused by CRISPR/Cas9 as well as other RNA-guided nucleases in living cells. Similar to LAM-PCR, it employs multiple PCRs to amplify regions of interest that contain a specific insert that preferentially integrates into double-stranded breaks. As gene therapy is an emerging field, GUIDE-Seq has gained traction as a cheap method to detect the off-target effects of potential therapeutics without needing whole genome sequencing.

References

  1. ↑ "insert - Terminology of Molecular Biology for insert – GenScript". www.genscript.com. Retrieved 22 October 2017.
  2. ↑ Hahne JC, Lampis A, Valeri N (February 2021). "Vault RNAs: hidden gems in RNA and protein regulation". Cellular and Molecular Life Sciences. 78 (4): 1487–1499. doi:10.1007/s00018-020-03675-9. PMC   7904556 . PMID   33063126.
  3. ↑ Levine B, Kroemer G (January 2019). "Biological Functions of Autophagy Genes: A Disease Perspective". Cell. 176 (1–2): 11–42. doi:10.1016/j.cell.2018.09.048. PMC   6347410 . PMID   30633901.
  4. ↑ "Herbert W. Boyer and Stanley N. Cohen". Science History Institute. 2016-06-01. Retrieved 2021-04-19.
  5. ↑ Malzahn A, Lowder L, Qi Y (2017-04-24). "Plant genome editing with TALEN and CRISPR". Cell & Bioscience. 7 (1): 21. doi:10.1186/s13578-017-0148-4. PMC   5404292 . PMID   28451378.
  6. ↑ Prill K, Dawson JF (2020). "Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?". Frontiers in Molecular Biosciences. 7: 595474. doi: 10.3389/fmolb.2020.595474 . PMC   7793982 . PMID   33425990.
  7. ↑ Mojica FJ, Rodriguez-Valera F (September 2016). "The discovery of CRISPR in archaea and bacteria". The FEBS Journal. 283 (17): 3162–9. doi:10.1111/febs.13766. hdl: 10045/57676 . PMID   27234458. S2CID   42827598.
  8. ↑ Barrangou R (February 2015). "The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology. 32: 36–41. doi:10.1016/j.coi.2014.12.008. PMID   25574773.
  9. ↑ Oh JH, van Pijkeren JP (2014-09-29). "CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri". Nucleic Acids Research. 42 (17): e131. doi:10.1093/nar/gku623. PMC   4176153 . PMID   25074379.
  10. ↑ Ishino Y, Krupovic M, Forterre P (April 2018). Margolin W (ed.). "History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology". Journal of Bacteriology. 200 (7): e00580–17, /jb/200/7/e00580–17.atom. doi:10.1128/JB.00580-17. PMC   5847661 . PMID   29358495.
  11. ↑ Ebrahimi V, Hashemi A (August 2020). "Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: A review". Gene. 753: 144813. doi:10.1016/j.gene.2020.144813. PMID   32470504. S2CID   219103770.
  12. ↑ Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR (2018-05-31). "Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template". Communications Biology. 1 (1): 54. doi:10.1038/s42003-018-0054-2. PMC   6123678 . PMID   30271937.
  13. ↑ Maganti HB, Bailey AJ, Kirkham AM, Shorr R, Pineault N, Allan DS (March 2021). "Persistence of CRISPR/Cas9 gene edited hematopoietic stem cells following transplantation: A systematic review and meta-analysis of preclinical studies". Stem Cells Translational Medicine. 10 (7): 996–1007. doi: 10.1002/sctm.20-0520 . PMC   8235122 . PMID   33666363.
  14. ↑ Bi H, Fei Q, Li R, Liu B, Xia R, Char SN, et al. (July 2020). "Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production". Plant Biotechnology Journal. 18 (7): 1526–1536. doi:10.1111/pbi.13315. PMC   7292542 . PMID   31821678.
  15. ↑ Charpentier E, Marraffini LA (June 2014). "Harnessing CRISPR-Cas9 immunity for genetic engineering". Current Opinion in Microbiology. 19: 114–119. doi:10.1016/j.mib.2014.07.001. PMC   4155128 . PMID   25048165.
  16. ↑ Boch J (February 2011). "TALEs of genome targeting". Nature Biotechnology. 29 (2): 135–6. doi:10.1038/nbt.1767. PMID   21301438. S2CID   304571.
  17. ↑ Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, et al. (July 2008). "Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification". Molecular Cell. 31 (2): 294–301. doi:10.1016/j.molcel.2008.06.016. PMC   2535758 . PMID   18657511.
  18. ↑ O'Brien JA, Lummis SC (June 2011). "Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles". BMC Biotechnology. 11 (1): 66. doi:10.1186/1472-6750-11-66. PMC   3144454 . PMID   21663596.
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