Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.     

Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms

Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.     

Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race

1. Download high-res image (273KB)
2. Download full-size image

     

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

1. Download : Download high-res image (90KB)
2. Download : Download full-size image

     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Frameshifting in Alphaviruses: A Diversity of 3′ Stimulatory Structures

Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal − 1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the − 1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein.The present work shows that there is remarkable diversity in the 3′ sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3′ RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3′-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of − 1 frameshifting stimulators in mammalian and insect systems.     

Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

Recent Advances in CRISPR‐Cas9 Genome Editing Technology for Biological and Biomedical Investigations

The Type II CRISPR‐Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single‐mutation genetic diseases, and multiple‐site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR‐Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81–94, 2018. © 2017 Wiley Periodicals, Inc.     

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

1. Download high-res image (150KB)
2. Download full-size image

     

基于CRISPR/Cas9技术的高通量筛选平台:发掘病毒复制相关宿主分子的新途径

病毒作为严格的细胞内寄生生物,需要多种宿主蛋白辅助其完成生命周期。寻找与病毒复制相关的宿主因子并揭示其作用机制,将有助于阐明病毒的感染机制,为疫病的防治提供新靶标。与RNA干扰技术相比,近年来兴起的CRISPR/Cas9技术能更特异、高效、准确地实现基因组编辑,因而在功能基因研究中得到更广泛应用。而基于CRISPR/Cas9系统的宿主全基因组sgRNA文库高通量筛选技术平台,可快速发现参与病毒侵入、复制等生物学过程的关键宿主因子,通过明确病毒-宿主分子相互作用进而揭示病毒的生命周期,为分子病毒学和免疫学提供了强大的研究工具。本文主要总结了基于CRISPR/Cas9技术的高通量筛选平台的具体筛选流程,归纳和讨论了该平台在筛选调控病毒复制相关宿主因子中的应用现状和发展前景。