TALENs (Transcription Activator-like Effector Nucleases)
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Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. TALENs are made by fusing a DNA-binding domain from TALE (transcription activator-like effector) proteins with a DNA cleavage domain from the FokI nuclease. This fusion allows TALENs to be targeted to specific DNA sequences and induce double-stranded breaks, making them useful for genome editing applications.
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Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
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Ppt of genome editing
This is a small presentation on Genome editing process. all techniques are described in short. Hope u all will like it.
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Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
Conventional techniques such as gene knockouts using P-elements and transposable genetic elements have been superseded by more accurate genome editing methods such as TALENs and CRISPR/Cas.
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Gene silencing is an epigenetic process that reduces or eliminates the production of a protein from its corresponding gene. There are two main types: transcriptional gene silencing via histone modifications, and post-transcriptional gene silencing by destroying or blocking mRNA. Methods to induce gene silencing include antisense oligonucleotides, ribozymes, and RNA interference using short interfering RNA or microRNA. RNAi leads to sequence-specific degradation of mRNA through the RNA-induced silencing complex. Gene silencing has applications in studying gene function, functional genomics, and medicine, with the goal of downregulating gene expression.
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CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.
Cas9 (CRISPR-associated protein 9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
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Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
Genome editing techniques
Genome editing techniques allow DNA to be inserted, deleted, modified or replaced in the genome of a living organism. Four families of engineered nucleases have been used for genome editing: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. Each system uses a different mechanism for recognizing and cutting DNA at specific locations to modify genes. The CRISPR/Cas9 system has become widely used due to its ease of design and lower off-target effects compared to other techniques.
Site specific recombination
This document discusses site-specific recombination, including the structures and mechanisms involved. It describes two classes of recombinases - tyrosine recombinases and serine recombinases. Tyrosine recombinases involve cleavage of DNA through formation of a protein-DNA bond using a tyrosine residue. Serine recombinases utilize a phosphoserine bond between DNA and a conserved serine residue. The document provides examples of applications for site-specific recombination such as tracking cell lineage, altering gene expression, and targeted gene knockout.
Protein dna interactions
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
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The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
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High throughput next generation sequencing and robust transcriptome analysis help with gene expression profiling, gene annotation or discovery of non-coding RNA.
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SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.
Rapid amplification of c-DNA ends
IT is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell
Site directed mutagenesis by pcr
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
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Physical mapping
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According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
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DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to cytosine residues in DNA. It is catalyzed by DNA methyltransferase enzymes and plays a key role in gene expression and cellular differentiation. Aberrant DNA methylation, including both hypermethylation and hypomethylation, has been associated with cancer development by disrupting gene expression. Detection of DNA methylation patterns can provide insights into cancer biology and may have applications as a diagnostic tool.
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TALENs (Transcription Activator-like Effector Nucleases)
- 1. (TALEN’s) Muhammad Mujahid ID: F2019253023
- 2. Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing of: i. DNA-binding domain (TAL effector). ii. DNA cleavage domain (a nuclease which cuts DNA strands).
- 3. TALEs • TALEs – Transcription Activator-Like Effector proteins produced by certain plant pathogenic bacteria, Xanthomonase spp. • Act as effectors during pathogenesis. • It contain: i. NLS (nuclear localization sequence). ii. N-terminal translocation signal. iii. Transcriptional activator domain. iv. Central repeat domain. • Contain 17 to 18 tandem repeats of a 33-35 amino acid motif that can recognise a single base through two di-variant residues •DNA binding specificity is determined by the amino acids at positions 12 and 13 within each repeat which is called Repeat Variable Diresidue (RVD) .
- 5. 5 Structure and DNA binding code of TALEs NG = T HD = C NI = A NN = G or A DNA binding code N’ C’ Central repeat domain ADNLS L T P E Q V V A I A S H D G G K Q A L E T V Q R L L P V L C Q A H G 1 3412 13 TS Repeats (1.5 to 33.5) Repeat variable diresidue (RVD) 12 13
- 6. 6 How to engineer TALEs? N’ TS C’ ADNLS HD HD HD NI NI NI NG NG NG NK NI NG HD NK DNA Target TCCCAAATTTGATCG Engineered central repeat domain = CHD NI NG NK = A = T = G
- 7. TALENs
- 8. G G A T G N N N N N N N N N N N N N N N N N N N C C T A C N N N N N N N N N N N N N N N N N N N 5’ 3’ 3’ 5’ Nuclease DNA-binding 21-Nov-16 8 Flavobacterium okeanokoites N-terminal: DNA-binding domain C-terminal: nonspecific DNA-cleavage activity
- 12. Transcription activator like Effector Nucleases (TALENs) is a synthetic modified Restriction Enzyme. It consist on TALEs (proteins) and Nuclease enzyme (Fok 1). It use as tool for Genome Editing. In 2011, named “The methods of the Year”
Editor's Notes
- Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501.