- Award ID(s):
- 1644965
- NSF-PAR ID:
- 10161890
- Date Published:
- Journal Name:
- Journal of visualized experiments
- Volume:
- 152
- ISSN:
- 1940-087X
- Page Range / eLocation ID:
- e60253
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or “basal” vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus , a small species of sturgeon. We introduced mutations into the genes Tyrosinase , which is needed for melanin production, and Sonic hedgehog , a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants’ various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.more » « less
-
Abstract Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9‐based genome editing platform inTragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 inTragopogon . We have established a feasible tissue culture and transformation protocol forTragopogon . Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted forTragopogon ) was capable of introducing site‐specific mutations inTragopogon protoplasts.Agrobacterium ‐mediated transformation with Cas9‐sgRNA constructs targeting the phytoene desaturase gene (TraPDS ) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles ofTraPDS in albino shoots fromTragopogon porrifolius (2x ) andTragopogon mirus (4x ), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9‐based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed inTragopogon , application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene‐balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems. -
Abstract Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Design and production of plasmids for base‐editing experimentsBasic Protocol 2 : Transfection of adherent cells and harvesting of genomic DNABasic Protocol 3 : Genotyping of harvested cells using Sanger sequencingAlternate Protocol 1 : Next‐generation sequencing to quantify base editingBasic Protocol 4 : Single‐cell isolation of base‐edited cells using FACSAlternate Protocol 2 : Single‐cell isolation of base‐edited cells using dilution platingBasic Protocol 5 : Clonal expansion to generate isogenic cell lines and genotyping of clones -
Abstract Understanding the evolutionary consequences of anthropogenic change is imperative for estimating long‐term species resilience. While contemporary genomic data can provide us with important insights into recent demographic histories, investigating past change using present genomic data alone has limitations. In comparison, temporal genomics studies, defined herein as those that incorporate time series genomic data, utilize museum collections and repeated field sampling to directly examine evolutionary change. As temporal genomics is applied to more systems, species and questions, best practices can be helpful guides to make the most efficient use of limited resources. Here, we conduct a systematic literature review to synthesize the effects of temporal genomics methodology on our ability to detect evolutionary changes. We focus on studies investigating recent change within the past 200 years, highlighting evolutionary processes that have occurred during the past two centuries of accelerated anthropogenic pressure. We first identify the most frequently studied taxa, systems, questions and drivers, before highlighting overlooked areas where further temporal genomic studies may be particularly enlightening. Then, we provide guidelines for future study and sample designs while identifying key considerations that may influence statistical and analytical power. Our aim is to provide recommendations to a broad array of researchers interested in using temporal genomics in their work.
-
Abstract CRISPR‐Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large‐scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA‐programmable nature of CRISPR‐Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR‐suppressor scanning. In a typical CRISPR‐suppressor scanning experiment, a pooled library of single‐guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ. This cellular pool of protein variants is then challenged with drug treatment to identify mutations conferring a fitness advantage. Drug‐resistance mutations identified with this approach can not only elucidate drug mechanism of action but also reveal deeper mechanistic insights into protein structure‐function relationships. In this article, we outline the framework for a standard CRISPR‐suppressor scanning experiment. Specifically, we provide instructions for the design and construction of a pooled sgRNA library, execution of a CRISPR‐suppressor scanning screen, and basic computational analysis of the resulting data. © 2022 Wiley Periodicals LLC.
Basic Protocol 1 : Design and generation of a pooled sgRNA librarySupport Protocol 1 : sgRNA library design using command‐line CRISPORSupport Protocol 2 : Production and titering of pooled sgRNA library lentivirusBasic Protocol 2 : Execution and analysis of a CRISPR‐suppressor scanning experiment