Тяжелые металлы в окружающей среде
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| Genome editing
Genome editing is a genetic manipulation approach in which DNA is inserted, removed or replaced at a precise location within the genome. Its application may involve the use of specialized engineered enzymes (nucleases), or may rely on recombination-based approaches. This new set of tools has made it possible to make (nearly) any modification we desire to any organism we wish. The origins of development of this new set of tools reside in the need to identify which gene as being involved in a disease or process. Historically two approaches have been utilized to this end: 1) Forward genetics (i.e. mutagenize a population of individuals or cells, than screen for phe- notype of interest) and 2) Reverse genetics (a gene is identified as potentially involved in dis- ease/process -> modify to analyze function). Although very potent, first genetic engineering tools (bacterial transformation, biolistics, and electroporation) have low levels of precision as they mainly rely on working with relatively large DNA molecules (genes, promotor regions, etc.). While compared to the classical breeding tools (where whole genomes are manipulated) these were considered major leap forward, the inherent randomness of integration of the desired DNA and difficulties at targeting specific sequences in the genome, thus hindering the knock-out options made them less than perfect for advanced genetic studies. The accumulating knowledge on the importance of not just the availability of given DNA sequence, but on its precise location and epigenetic modification, accompanied by low success rate of the above ―classical‖ engineering tools made the need of developing the next generation of preci- sion instruments ever more pressing. This was further aggravated by the accumulation of data from genome-wide association studies (GWAS) where hundreds and thousands of single-nucleotide vari- ations in the genome are tagged as potentially affecting given trait. Verification of their function is possible only through the reverse genetic approach. That means such single nucleotide polymor- phisms (SNPs) need to be modified so that their effects can be revealed. As the first generation of genetic engineering tools was virtually incapable of fulfilling this task, a new set of tools was pro- posed that allow for precise alterations in the genome. Two main approaches are currently employed in this respect: 1) Recombination-based and 2) utilization of engineered nucleases. One of the first such tools used some viral genome editing mechanisms. Recombinant Adeno-Associated Virus (or rAAV) uses an exchange of nucleotide se- quences to enable insertion, deletion or replacement of DNA sequences in cells. This is achieved through homologous recombination (HR) between AAV particle and the target DNA, thus allowing for precise and specific editting-on-demand the genome of any mammalian cell down to single base-pair resolution and without sequence error. In the second approach some nucleases were engineered so that they cut at a desired position in the genome. First type of such enzymes were a group of Zinc Finger Nucleases (ZFNs) – engi- neered DNA binding proteins that can be designed to bind to a wide variety of DNA sequences and function by introducing a double stranded break at a specified location in the genome. ZFNs are comprised of two component parts: DNA-binding domain – made up of a chain of two-finger modules, each recog- nizing a unique hexamer (6 bp) sequence of DNA. Two-finger modules are stitched together to form a Zinc Finger Protein, with specificity of ≥ 24 bp DNA-cleaving domain - nuclease domain of Fok1 68 69 A high level of specificity is achieved with ZFNs because the Fok1 must dimerize for cleav- age to occur. As such, a pair of ZFNs must be designed (one each side of the cut site on opposite strands) and only when dual binding occurs does dimerization and cleavage ensue. Once the cut has been achieved, the cell activates repair pathways to fix the damage. The non-homologous end join- ing (NHEJ) pathway is error-prone, and this can be particularly useful to introduce disruptions in the coding region of a gene. Small insertions and deletions are commonly introduced at the cut site, often resulting in disruption of the coding frame of an associated gene. ZFNs are therefore particu- larly effective at knocking out gene function. A modification of this approach is the use of transcription activator-like effector nucleases (TALENs). TALENs are made by fusing a group of DNA-binding domains to a DNA cleavage do- main. 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. Studying the acquired immunity in bacteria yet another nuclease was identified that ap- peared capable of cutting at precise location. As these nucleases were initially found to be associat- ed to some Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) they were named Cas (CRISPR-associated) proteins. Several such proteins were identified (of which Cas-9 is the most widely utilized) the activity of which was later found to be triggered by the presence of a specific small RNAs, called small guiding RNAs (sgRNAs). As these RNAs are quite short creating them appeared very simple and this made this entire system very easy to use. Another very useful property of the system is that the sgRNA can bind on either strand of DNA and the Cas9 will cleave both strands (double strand break, DSB). Such DSB results in the silencing of that DNA sequence and allows for either removing or inserting specific nucleotides at the site of cleavage. Changing the target specificity of the RNA-protein complex does not require protein engineering but only the design of the short crRNA guide. Figure. Genomic manipulation with the CRISPR-Cas system requires only the Cas9 protein and an engineered small guide RNA (sgRNA) with a protospacer associated motif (PAM) sequence upstream of target complementary sequence. Owing to this ease of customization, CRISPR–Cas9 is now used to edit or modify the ge- nomes of a vast number of cells and organisms, including bacteria, parasites, plants, zebrafish, mice and human cells. When co-expressed with custom-designed sgRNAs in cells, Cas9 generates double strand brakes (DSBs) in genomic DNA that are subsequently repaired by non-homologous end- joining (NHEJ) to introduce gene disruptions or by homology directed repair (HDR) through the insertion of donor genetic sequences. Introducing DSBs at defined positions can generate cell lines carrying chromosomal translocations that resemble those that occur in naturally observed cell line- ages. In addition, the targeting of several loci simultaneously with multiple sgRNAs has also been achieved, which is known as multiplexing. CRISPR–Cas9 thus provides a robust and malleable tool to study genomic rearrangements, and increases our understanding of the development of different tolerance mechanisms. A modification of this system uses novel non-Cas9 CRISPR system isolated from Francisel- жүктеу/скачать 6.49 Mb. Достарыңызбен бөлісу:
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