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Published in Michael Hehenberger, Zhi Xia, Huanming Yang, Our Animal Connection, 2020
Michael Hehenberger, Zhi Xia, Huanming Yang
To understand how knockout mice are created, we need to go back to the role played by DNA in carrying information about the development and function of our bodies throughout life. Our DNA is packaged in chromosomes, which occur in pairs—one inherited from the father and the other from the mother. Exchange of DNA sequences within such chromosome pairs increases genetic variation in the population and occurs by a process called homologous recombination. Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is used by our cells to accurately repair harmful breaks that occur on both strands of DNA. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. The 2007 Nobel Laureates Mario Capecchi and Oliver Smithies first demonstrated that homologous recombination could be used to specifically modify genes in mammalian cells.
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Published in Michael Hehenberger, Zhi Xia, Our Animal Connection, 2019
To understand how knockout mice are created, we need to go back to the role played by DNA in carrying information about the development and function of our bodies throughout life. Our DNA is packaged in chromosomes, which occur in pairs—one inherited from the father and the other from the mother. Exchange of DNA sequences within such chromosome pairs increases genetic variation in the population and occurs by a process called homologous recombination. Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is used by our cells to accurately repair harmful breaks that occur on both strands of DNA. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. The 2007 Nobel Laureates Mario Capecchi and Oliver Smithies first demonstrated that homologous recombination could be used to specifically modify genes in mammalian cells.
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Homologous recombination techniques are based on high sequence homology of the parental gene sequences. The resultant chimeric gene shows an amalgamation of characteristic features of the combining parental sequences. The homologous recombination methods have been classified into in vitro and in vivo homologous recombination methods. DNA shuffling is the most prominent in vitro homologous recombination technique developed by Stemmer and coworkers (Stemmer, 1994b). Later advancements in the DNA shuffling method resulted in improvised schemes such as family shuffling (Crameri et al., 1998), and DOGs (degenerate oligonucleotide gene shuffling) (Gibbs et al., 2001). Other in vitro homologous recombination methods include: random priming in vitro recombination (RPR) (Shao et al., 1998), truncated metagenomic gene-specific PCR (TMGS-PCR) (Wang et al., 2010), staggered extension process (StEP) (Zhao et al., 1998), random chimeragenesis on transient templates (RACHITT) (Coco, 2003), synthetic shuffling (Ness et al., 2002). The in vivo homologous recombination methods include: cloning performed in yeast (CLERY) (Abecassis et al., 2003), Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) (Gonzalez-Perez et al., 2014), and phage assisted continuous evolution (PACE) (Esvelt et al., 2011).
Predicting algorithm of attC site based on combination optimization strategy
Published in Connection Science, 2022
Zhendong Liu, Xi Chen, Dongyan Li, Xinrong Lv, Mengying Qin, Ke Bai, Zhiqiang He, Yurong Yang, Xiaofeng Li, Qionghai Dai
Gene recombination is a way that organisms use recombinase to recombine different genes to produce new genotype individuals. It is widely present in prokaryotes and has important meanings such as maintaining biological genetic diversity and promoting biological evolution (Epum & Haber, 2022). Common recombination includes: homologous recombination, translocation recombination and site-specific recombination. Currently, with the development of site-specific recombination systems, site-specific recombination technology has been extensively used in various biological genetic engineering operations, especially in higher eukaryotes (Bessen et al., 2019; Häcker et al., 2017). Site-specific recombination refers to the integration, excision and transformation of DNA fragments between specific sites, which is catalysed by integron integrase. This type of recombination is associated with specific DNA sequences in bacteriophages and bacteria, and the reaction always involves two DNA-specific sites. However, these two specific sites usually have very similar or even exactly the same DNA sequences. Such sequence-level constraints restrict efficient recombination between the two sites (Tian & Zhou, 2021). Therefore, in order to solve the problem of sequence constraints, it is necessary to study the structure of specific recombination sites in the recombination system.
Development of capability for genome-scale CRISPR-Cas9 knockout screens in New Zealand
Published in Journal of the Royal Society of New Zealand, 2018
Francis W. Hunter, Peter Tsai, Purvi M. Kakadia, Stefan K. Bohlander, Cristin G. Print, William R. Wilson
Although evolution has selected CRISPR-Cas systems for protection against transfer of potentially pathogenic nucleic acids, the re-engineered systems have now acquired very different biological roles including dissecting the genetics of complex organisms through precision gene editing of germline cells (Doudna & Sternberg 2017), the latter constituting a new, directed evolutionary role. As a precision mutagen, CRISPR-Cas9 recruits endogenous DNA double-strand break repair pathways in cells to effect quite different outcomes. Thus, in the presence of an exogenous donor template with homology to sequences flanking the cut site, high-fidelity homologous recombination repair (HRR) can replace the DNA sequence between the flanking homology arms with an alternate sequence. In the absence of such a template, or in non-proliferating cells lacking HRR competence, repair proceeds primarily via error-prone pathways such as non-homologous end-joining or microhomology-mediated end-joining. These ‘emergency’ repair pathways restore chromosomal integrity by repairing breaks at all costs—the major cost being the introduction of small insertion/deletion mutations at the cut site (Figure 2). These are typically frameshifting; thus, targeting the first conserved exon of a gene is an efficient and reliable way of generating knockout mutations that ablate gene function. More recently, the fundamental action of CRISPR systems—targeting an effector protein to specific genetic loci—has been exploited to develop a range of useful variations of the CRISPR-Cas9 tool (Figure 3). For example, Cas9 has been rationally engineered by site-directed mutagenesis or the addition of fusion proteins to produce variants with altered catalytic activity, enhanced sequence specificity or that tag, isolate, epigenetically suppress or even activate target genes (Figure 3).