Genetics O Level Biology Notes Pdf

[Curtis Cassel]

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Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or 100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.

Systems Biology is a natural extension of molecular and cellular biology,1,2,3 which consists of multi-stage processes beginning with a (1) comprehensive identification and (2) quantitative analysis of individual system components and their networked interaction, which leads to the ability to (3) control existing systems toward the desired state and (4) design new systems based on an understanding of the underlying structural and dynamical principles. After identification of key genes by classical forward and reverse genetics, systems biology in mammals has been further accelerated by a series of genome projects, especially at the molecular-to-cellular levels, where in vitro cell culture systems allow system-level identification, analysis, control, and design of molecular networks. On the other hand, organism-level systems biology in mammals still remains an important challenge in biology.4

Mammalian genetics (particularly in mice) has been widely exploited in order to investigate complex and dynamic biological processes executed by molecular networks and/or cellular circuits in organisms. Forward genetics (germline mutagenesis and gene-trap) and reverse genetics (targeted KO or KI) are available in mouse genetics as in other model organisms such as yeast, nematode and fly. Especially, developmental engineering based on the establishment of cultured ESCs was often used to generate KO and/or KI mice.5,6,7 Various genetic tools can be also introduced by transgenic (Tg) mice techniques.8

Conventional and next-generation mammalian genetics. a A typical procedure for conventional mouse genetics. Upper panel: generation of a transgenic mouse, lower panel: gene targeting in ESCs and generation of the mutated mouse. An inbred strain such as C57BL/6 (B6) is widely used for final analysis, while hybrid or other inbred strains are used in the production stages for practical reasons. Therefore, a prolonged backcross procedure is needed in many cases. In addition, gene targeting in ESCs is dependent on a spontaneous DSB and following HDR in the cells, causing an inefficient targeting rate. b In next-generation mouse genetics, all of the crossing procedures are not needed because of the use of an inbred strain for analysis, efficient genome editing in zygotes or ESCs mediated by site-specific endonucleases, and one-step generation of the genome-edited bi-allelic KO mouse or KI ES mouse. These F0 animals can be used in subsequent phenotyping experiments

Despite the limitations of conventional mammalian genetics, systematic, large-scale mouse genetics projects have been performed. For example, ethyl-nitrosourea mutagenesis in mice was exploited to screen mammalian circadian clock genes10,11,12 and for systematic gene function studies.13, 14 The gene-trap strategy has more recently been applied to such forward-genetics approaches, and >100,000 of trapped ESC lines have been established and kept in international organizations (e.g., International gene trap consortium or IGTC, ).15 Other systematic international efforts to collect, prepare and maintain mutant mice and ESCs have also been performed, such as the International Knockout Mouse Consortium/International Mouse Phenotype Consortium ( ).16,17,18,19 Multiple Cre Tg/KI strains have also been established by individual researchers, institutes and international consortiums.20,21,22 However, to carry out organism-level systems biology, these large-scale efforts should be scaled down to the single-laboratory scale or even to the single-researcher scale. To address this technological challenge, next-generation mammalian genetics without crossing is proposed here to allow completion of KO or KI mouse production and phenotyping analysis within the F0 generation (Fig. 1b). This can be realized by the application of highly efficient genome editing by site-specific nucleases for one-step generation of whole-body genome-edited inbred animals within a single generation. Recently, there has been rapid progress in next-generation mammalian genetics, as introduced below, which will form an essential platform for organism-level systems biology.

Three major classes of site-specific endonucleases have been used for genome-editing,27 zinc-finger nuclease (ZFN),28 transcription activator-like effector nuclease (TALEN)29 and clustered regularly interspaced short palindromic repeats (CRISPR)-associated Protein9 (Cas9).30 ZFN and TALEN are categorized into customizable endonucleases because they are composed of a customizable sequence-specific DNA-binding domain fused to a nonspecific DNA catalytic domain of FokI endonuclease.31 On the other hand, Cas9 is a RNA-guided endonuclease and recruited to specific DNA sequences by a short RNA guide molecule that recognizes target DNA via base-pairing.32

Dimerization of the FokI endonuclease catalytic domain is essential for cleavage of DNA by ZFN and TALEN.31 This means that two ZFN or TALEN molecules must bind on both right and left sides of the target site with an appropriate orientation and spacing. Therefore, the dimer recognizes 2-fold longer sequence at the target site than single ZFN or TALEN molecules. This molecular property gives higher specificity and reduced off-target effect.

Targeted insertion or KI of a DNA fragment with mutated sequence, short functional sequence (restriction enzyme site, recombinase recognition site, or protein tag etc.), or functional expression cassette can be also facilitated via HDR, NHEJ and MMEJ by co-transfer of linear or circular donor vector, PCR fragment or single-stranded oligo DNA nucleotide (ssODN) together with the site-specific endonucleases (Fig. 2).

NHEJ-mediated fragment insertion/KI is easier and more efficient than the HR pathway, because the NHEJ-repair reaction is thought to predominate over the HR reaction for DSB repair.76, 77 In the NHEJ-mediated insertion, both the donor plasmid and the target genome loci are digested simultaneously. And then, the digested donor plasmid is integrated into the digested genome loci. A PCR fragment or double-stranded ODN can be also applied as an integrated fragment without digestion. This pathway works not only in cultured mammalian cells (including ESCs) but also in zebrafish, and does not necessarily require antibiotic selection.56, 78,79,80,81 In addition, there is no need to prepare a targeting vector with long homology arms, which is generally a time-consuming process. On the other hand, it is of note that the direction of the inserted fragment is not controllable, and indels are usually introduced at the junction site. Therefore, the method is inappropriate for some KI purposes, such as in-frame KI of an exogenous ORF into an endogenous gene.

The compelling advantages of the site-specific endonucleases in efficient genome-editing has been examined in recent years. In particular, zygotic genome editing enables one-step production of genome-edited animals, skipping the in vitro targeting step in ESCs. Introduction of components into one-cell zygotes are relatively simple and easy, particularly for the CRISPR/Cas9 system, which just requires cytoplasmic microinjection or electroporation.84,85,86,87

Validity of ES mouse production and phenotyping analysis within a single generation was first proposed and tested by using the tetraploid complementation method.106,107,108,109,110,111,112,113 However, several possible drawbacks of the method are known. First, substantial contamination of host cells was often observed in chimera mice produced by this method, which can cause developmental abnormalities.113,114,115 Second, the genetic heterozygosity of both tetraploid embryos and ESCs seems to be crucial for survival of the resultant ES mice,110, 112, 114 which means that the use of inbred ESCs does not seem possible and further backcrossing is required. In addition, preparing hundreds of tetraploid embryos every time does not seem practical for routine generation of many ES mice. For these reasons, few reports have used tetraploid complementation in a large-scale phenotyping assay of ES mice.

As discussed above, high-throughput KO or KI mouse production is pivotal for accelerating system-level identification, and analysis of molecular networks and cellular circuits in organisms. Given that various genetic tools, such as optogenetics and chemogenetic tools160, 161 are developing rapidly in recent years, high-throughput genome-edited mouse production is required for their in vivo implementation. Next-generation mammalian genetics potentially enables a single laboratory or a single researcher to generate, maintain and analyze multiple genome-edited strains rather than institutes or consortiums for production, deposit and distribution of various strains and ESCs. Because sgRNAs for targeted sites can be readily designed and prepared, the CRISPR/Cas9 system, in particular, makes such large-scale genetics feasible. Indeed, a recent study generated 31 novel CRISPR-KO mice lacking testis-expressing genes.162 Since whole-body bi-allelic KO rates were not sufficiently high for the next-generation scheme, the authors performed the crossing of selected F0 founders based on sequence and PCR screening data. In order to realize almost perfect (100%) whole-body bi-allelic KO rate for next-generation mammalian genetics, we recently performed triple-CRISPR-based large-scale reverse genetics for sleep research. To identify genes involving neural electrophysiological activities during sleep or wake, we first developed an average neuron model in silico and found that genes involved in intracellular Ca2+ regulation (Ca2+ channels, Ca2+-dependent channels, Ca2+-pumps or Ca2+-dependent enzymes) are important for electrophysiological slow-wave-oscillation patterns during sleep. To further assess the roles of these genes in vivo, we next produced KO mice for 33 genes with the triple-CRISPR methods and eventually identified 8 genes important for regulating sleep duration.62, 163

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