279:) to target specific genes. TALENs are nucleases that have two important functional components: a DNA binding domain and a DNA cleaving domain. The DNA binding domain is a sequence-specific transcription activator-like effector sequence while the DNA cleaving domain originates from a bacterial endonuclease and is non-specific. TALENs can be designed to cleave a sequence specified by the sequence of the transcription activator-like effector portion of the construct. Once designed, a TALEN is introduced into a cell as a plasmid or mRNA. The TALEN is expressed, localizes to its target sequence, and cleaves a specific site. After cleavage of the target DNA sequence by the TALEN, the cell uses non-homologous end joining as a DNA repair mechanism to correct the cleavage. The cell's attempt at repairing the cleaved sequence can render the encoded protein non-functional, as this repair mechanism introduces insertion or deletion errors at the repaired site.
257:. CRISPR-associated (cas) genes encode cellular machinery that cuts exogenous DNA into small fragments and inserts them into a CRISPR repeat locus. When this CRISPR region of DNA is expressed by the cell, the small RNAs produced from the exogenous DNA inserts serve as a template sequence that other Cas proteins use to silence this same exogenous sequence. The transcripts of the short exogenous sequences are used as a guide to silence these foreign DNA when they are present in the cell. This serves as a kind of acquired immunity, and this process is like a prokaryotic RNA interference mechanism. The CRISPR repeats are conserved amongst many species and have been demonstrated to be usable in human cells, bacteria,
152:(RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (
197:
183:, or other applications. RNA interference is a very useful research tool, allowing investigators to carry out large genetic screens in an effort to identify targets for further research related to a particular pathway, drug, or phenotype.
156:). The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.
74:
In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of
267:, and other organisms for effective genome manipulation. The use of CRISPRs as a versatile research tool can be illustrated by many studies making use of it to generate organisms with genome alterations.
179:
give laboratories a way of testing many genes in a variety of experimental backgrounds. Insights gained from experimental RNAi use may be useful in identifying potential therapeutic targets,
599:
Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J (November 2000). "Functional genomic analysis of C. elegans chromosome I by systematic RNA interference".
497:
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (February 1998). "Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans".
1015:
Gennequin B, Otte DM, Zimmer A (November 2013). "CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene".
129:. Researchers draw inferences from how the knockdown differs from individuals in which the gene of interest is operational. Transient knockdowns are often used in
276:
207:
775:
Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (July 2013).
336:
Summerton JE (2007). "Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity".
71:, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result is referred to as a "transient knockdown".
1156:
1050:
Sun N, Zhao H (July 2013). "Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing".
287:
So far, knockdown organisms with permanent alterations in their DNA have been engineered chiefly for research purposes. Also known simply as
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176:
236:
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Another technology made possible by prokaryotic genome manipulation is the use of transcription activator-like effector nucleases (
153:
1161:
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Summerton J (December 1999). "Morpholino antisense oligomers: the case for an RNase H-independent structural type".
55:
of an organism is genetically modified, the resulting organism is called a "knockdown organism." If the change in
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825:
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161:
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RNAi is widely used as a laboratory technique for genetic functional analysis. RNAi in organisms such as
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95:
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is a mechanism involving loci called 'Clustered
Regularly Interspaced Short Palindromic Repeats', or
32:
218:
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632:
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444:
361:
302:
There are several companies that offer commercial services related to gene knockdown treatments.
927:"Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination"
462:
Wahlestedt, Claes (February 1994). "Antisense oligonucleotide strategies in neuropharmacology".
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Kamath RS, Ahringer J (August 2003). "Genome-wide RNAi screening in
Caenorhabditis elegans".
291:, these organisms are most commonly used for reverse genetics, especially in species such as
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125:, but has an unknown or incompletely known function. This experimental approach is known as
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Nasevicius A, Ekker SC (October 2000). "Effective targeted gene 'knockdown' in zebrafish".
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Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM (November 2013).
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777:"CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes"
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development. The term gene knockdown first appeared in the literature in 1994
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provides a quick and inexpensive means of investigating gene function. In
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A different means of silencing exogenous DNA that has been discovered in
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24:
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that has a sequence complementary to either gene or an mRNA transcript.
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550:"The RNA-induced silencing complex: a versatile gene-silencing machine"
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and will be present in the daughter cells of the injected cell through
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cleavage sites used for maturation of other functional RNAs, including
36:
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976:"Genome editing using artificial site-specific nucleases in zebrafish"
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Ghadakzadeh S, Mekhail M, Aoude A, Hamdy R, Tabrizian M (March 2016).
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826:"Orthogonal Cas9 proteins for RNA-guided gene regulation and editing"
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for which transient knockdown technologies cannot easily be applied.
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The most direct use of transient knockdowns is for learning about a
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Biochimica et
Biophysica Acta (BBA) - Gene Structure and Expression
878:"RNA-guided editing of bacterial genomes using CRISPR-Cas systems"
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736:"Small Players Ruling the Hard Game: siRNA in Bone Regeneration"
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Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (March 2013).
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91:-H dependent antisense, or through the blocking of either
652:"RNAi therapeutics: principles, prospects and challenges"
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79:(in the case of gene-binding), the degradation of the
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31:is reduced. The reduction can occur either through
133:because oligos can be injected into single-celled
175:research, the availability of tools such as the
114:oligos or other RNase-H independent antisense).
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8:
974:Hisano Y, Ota S, Kawahara A (January 2014).
925:Chen C, Fenk LA, de Bono M (November 2013).
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83:transcript (e.g. by small interfering RNA (
19:is an experimental technique by which the
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980:Development, Growth & Differentiation
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237:Learn how and when to remove this message
1140:"TALEN Effector Nucleotide Targeter 2.0"
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208:not related to the topic of the article
1124:"CRISPR Genome Engineering Resources"
338:Current Topics in Medicinal Chemistry
7:
740:Journal of Bone and Mineral Research
1132:"Mojo Hand: Design Your Own TALENs"
554:The Journal of Biological Chemistry
650:Aagaard L, Rossi JJ (March 2007).
14:
1094:"Adenoviral Gene Knockdown Cells"
548:Pratt AJ, MacRae IJ (July 2009).
1096:. Sirion Biotech. Archived from
1052:Biotechnology and Bioengineering
195:
1157:Genetically modified organisms
656:Advanced Drug Delivery Reviews
1:
713:10.1016/S1046-2023(03)00050-1
393:10.1016/S0167-4781(99)00150-5
217:or discuss this issue on the
476:10.1016/0165-6147(94)90107-4
67:or temporarily binding to a
39:such as a short DNA or RNA
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1029:10.1016/j.bbrc.2013.10.138
793:10.1016/j.cell.2013.06.044
668:10.1016/j.addr.2007.03.005
350:10.2174/156802607780487740
47:Versus transient knockdown
168:Drosophila melanogaster
35:or by treatment with a
931:Nucleic Acids Research
567:10.1074/jbc.R900012200
1142:. Cornell University.
206:may contain material
177:Ahringer RNAi Library
131:developmental biology
23:of one or more of an
882:Nature Biotechnology
464:Trends Pharmacol Sci
215:improve this section
33:genetic modification
1162:Genetics techniques
613:2000Natur.408..325F
511:1998Natur.391..806F
1134:. The Mayo Clinic.
1126:. Broad Institute.
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845:10.1038/nmeth.2681
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100:RNA splicing
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470:(2): 42–6.
251:prokaryotes
96:translation
1151:Categories
318:References
289:knockdowns
260:C. elegans
227:April 2020
173:C. elegans
162:C. elegans
112:morpholino
102:sites, or
21:expression
265:zebrafish
219:talk page
139:embryonic
123:sequenced
110:(e.g. by
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358:17430206
306:See also
104:nuclease
25:organism
1080:6214542
952:3814388
903:3748948
854:3844869
802:3770145
701:Methods
677:1978219
637:4373444
609:Bibcode
577:2709356
535:4355692
527:9486653
507:Bibcode
484:8165722
255:CRISPRs
187:CRISPRs
135:zygotes
98:, pre-m
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271:TALENs
87:)) or
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362:S2CID
108:miRNA
89:RNase
85:siRNA
51:If a
29:genes
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859:PMID
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781:Cell
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385:1489
354:PMID
297:rats
293:mice
165:and
154:RISC
119:gene
93:mRNA
81:mRNA
69:gene
65:mRNA
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