Insert (molecular biology)

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]

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.

## Contents

* 1 History
* 2 Techniques and protocols
* 3 CRISPR gene editing
* 4 Transcription activator-like effector nuclease
* 5 Zinc finger nuclease
* 6 Gene gun
* 7 References

## 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]

## References

1. ↑ "insert - Terminology of Molecular Biology for insert – GenScript". https://www.genscript.com/molecular-biology-glossary/10173/insert.
2. ↑ "Vault RNAs: hidden gems in RNA and protein regulation". Cellular and Molecular Life Sciences 78 (4): 1487–1499. February 2021. doi:10.1007/s00018-020-03675-9. PMID 33063126.
3. ↑ "Biological Functions of Autophagy Genes: A Disease Perspective". Cell 176 (1–2): 11–42. January 2019. doi:10.1016/j.cell.2018.09.048. PMID 30633901.
4. ↑ "Herbert W. Boyer and Stanley N. Cohen" (in en). 2016-06-01. https://www.sciencehistory.org/historical-profile/herbert-w-boyer-and-stanley-n-cohen.
5. ↑ "Plant genome editing with TALEN and CRISPR". Cell & Bioscience 7 (1): 21. 2017-04-24. doi:10.1186/s13578-017-0148-4. PMID 28451378.
6. ↑ "Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?" (in English). Frontiers in Molecular Biosciences 7: 595474. 2020. doi:10.3389/fmolb.2020.595474. PMID 33425990.
7. ↑ "The discovery of CRISPR in archaea and bacteria". The FEBS Journal 283 (17): 3162–9. September 2016. doi:10.1111/febs.13766. PMID 27234458.
8. ↑ "The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology 32: 36–41. February 2015. doi:10.1016/j.coi.2014.12.008. PMID 25574773.
9. ↑ "CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri". Nucleic Acids Research 42 (17): e131. 2014-09-29. doi:10.1093/nar/gku623. PMID 25074379.
10. ↑ "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. April 2018. doi:10.1128/JB.00580-17. PMID 29358495.
11. ↑ "Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: A review". Gene 753: 144813. August 2020. doi:10.1016/j.gene.2020.144813. PMID 32470504.
12. ↑ "Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template". Communications Biology 1 (1): 54. 2018-05-31. doi:10.1038/s42003-018-0054-2. PMID 30271937.
13. ↑ "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. March 2021. doi:10.1002/sctm.20-0520. PMID 33666363.
14. ↑ "Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production". Plant Biotechnology Journal 18 (7): 1526–1536. July 2020. doi:10.1111/pbi.13315. PMID 31821678.
15. ↑ "Harnessing CRISPR-Cas9 immunity for genetic engineering". Current Opinion in Microbiology 19: 114–119. June 2014. doi:10.1016/j.mib.2014.07.001. PMID 25048165.
16. ↑ "TALEs of genome targeting". Nature Biotechnology 29 (2): 135–6. February 2011. doi:10.1038/nbt.1767. PMID 21301438.
17. ↑ "Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification". Molecular Cell 31 (2): 294–301. July 2008. doi:10.1016/j.molcel.2008.06.016. PMID 18657511.
18. ↑ "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. June 2011. doi:10.1186/1472-6750-11-66. PMID 21663596.

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