Deep molluscan phylogeny: synthesis of palaeontological and neontological data

The position of the earliest-derived living molluscs, the Polyplacophora (chitons) and shell-less vermiform Aplacophora, remains highly contentious despite many morphological, developmental and molecular studies of extant organisms. These two groups are thought to represent either a basal molluscan grade or a clade (Aculifera) sister to the 'higher' molluscs (Conchifera). These incompatible hypotheses result in very different predictions about the earliest molluscs. A new cladistic analysis incorporating both Palaeozoic and extant molluscs is presented here. Our results support the monophyly of Aculifera and suggest that extant aplacophorans and polyplacophorans both derive from a disparate group of multivalved molluscs in two major clades. Reanalysis of the critical Ordovician taxon 'Helminthochiton' thraivensis shows that this animal lacks a true foot despite bearing polyplacophoran-like valves. Its position within our phylogenetic reconstruction indicates that many fossil 'polyplacophorans' in the order Palaeoloricata are likely to represent footless stem-group aplacophorans. 'H.' thraivensis and similar forms such as Acaenoplax may be morphological stepping stones between chitons and the shell-less aplacophorans. Our results imply that crown-group molluscan synapomorphies include serial repetition, the presence of a foot, a mineralized scleritome and a creeping rather than worm-like mode of life.     

The origins of molluscs

The interrelationships and evolutionary history of molluscs have seen great advances in the last decade. Recent phylogenetic studies have allowed alternative morphology‐based evolutionary scenarios to be tested and, most significantly, shown that the aplacophorans are sister group to polyplacophorans (chitons), corroborating palaeontological and embryological evolutionary scenarios in which aplacophorans are secondarily simplified from a chiton‐like ancestor. Aplacophoran morphology therefore does not represent the plesiomorphic condition for molluscs as a whole. The mollusc crown group radiated in the Early Cambrian, and rapidly thereafter, stem lineages to the major molluscan classes emerged: cephalopods, gastropods, bivalves (= pelecypods), monoplacophorans, rostroconchs (inferred stem scaphopods) and aculiferans. This attests to the fast, adaptive radiation of the crown group during the Cambrian explosion. Kimberella from the latest Ediacaran exhibits several molluscan traits, which justifies its position as a molluscan stem‐group member, rather than as a more basal Lophotrochozoan. The interrelationships among the conchiferan molluscs are still a matter of contention and require further palaeontological and molecular phylogenetic scrutiny.     

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated nuclease 9(Cas9)-based genome editing tool pCas/pTargetF system that we establi...     

Gene editing and its applications in biomedicine

Science China Life Sciences - The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and...     

基于CRISPR/Cas体系的生物传感器在病原体核酸检测方面的应用

成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas) 因高效的靶向结合和可编程性,已被开发为一种精准、高效、低价和高灵敏度的核酸检测工具。目前基于CRISPR-Cas体系的生物传感器在病原体核酸检测方面显示出了优良的性能,受到了人们的广泛关注,这种新型的病原体核酸检测有望替代传统的病原体检测方法。文中就基于CRISPR/Cas体系的生物传感器在病原体核酸检测中的最新研究进展进行综述。     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea

Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.     

Experimental Definition of a Clustered Regularly Interspaced Short Palindromic Duplicon in Escherichia coli

We analyzed sequences of newly inserted repeats in an Escherichia coli CRISPR (clustered regularly interspaced short palindromic repeats) array in vivo and showed that a base previously thought to belong to the repeat is actually derived from a protospacer. Based on further experimental results, we propose to use the term "duplicon" for a repeated sequence in a CRISPR array that serves as a template for a new duplicon. Our findings suggest the possibility of redrawing the borders between repeats, spacers, and protospacer adjacent motifs.     

Connecting genotypes,phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR‐associated nuclease 9) genome‐editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the genome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long‐standing goal of connecting genotypes, phenotypes and fitness.     

CRISPR adaptive immunity systems of prokaryotes

CRISPR (clustered regularly interspaced short palindromic repeats) systems were found in the genomes of many eubacteria and the vast majority of archaea. Their unique feature is that CRISPR-mediated immunity is adaptive and can be programmed to protect cells against mobile genetic elements. The review describes the general principles of CRISPR-mediated immunity and characterizes the CRISPR systems of various microorganisms.     

CRISPR enables heritable genome editing in planta

Recent exciting developments in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing showcase its potential to rapidly and efficiently edit genomes in planta, eliminating long processes of tissue culture and extensive breeding for crop improvement. These new methods offer heritable transgene-free edits in one generation, making them an attractive option for improving commercially important crops.     

CRISPR-based point-of-care plant disease diagnostics

The coupling of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas RNA-programmable nucleases with nucleic acid detection platforms has brought radical changes to the field of disease diagnosis. Recently, Sánchez et al. developed a simple, rapid, highly sensitive, precise, and in-field deployable point-of-care (POC) and point-of-need (PON) molecular disease detection tool that can be used in diverse agricultural applications.     

CRISPR/Cas in Arabidopsis: overcoming challenges to accelerate improvements in crop photosynthetic efficiencies

The rapid and widespread adoption of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technologies has allowed genetic editing in plants to enter a revolutionary new era. In this mini review, we highlight the current CRISPR/Cas tools available in plants and the use of Arabidopsis thaliana as a model to guide future improvements in crop yields, such as enhancing photosynthetic potential. We also outline the current socio‐political landscape for CRISPR/Cas research and highlight the growing need for governments to better facilitate research into plant genetic‐editing technologies.     

Comparison of CRISPR/Cas and Argonaute for nucleic acid tests

Guided, programmable, and target-activated nucleases, exemplified by Cas in the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and Argonaute (Ago), are emerging as a new generation of nucleic acid tests (NATs). A specific approach for comparison of these two nucleases side by side in terms of similarities, differences, and complementarities is instrumental for the sensible design of novel NATs.     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses

Recent experimental evidence has demonstrated that bacteria acquire resistance to viruses by incorporation of short transcribed nucleotide sequences into regions of clustered regularly interspaced short palindromic repeats (CRISPR). We have analysed community genomic data from acidophilic microbial biofilms and discovered that evolution of the CRISPR regions in two distinct Leptospirillum group II bacteria occurs fast enough to promote individuality in otherwise nearly clonal populations. Comparative genomics strongly indicates very recent lateral transfer of the CRISPR locus between these populations, followed by significant loss of spacer sequences and locus expansion by unidirectional heterogeneous addition of new spacer sequences. Diversification of the CRISPR region is inferred to be a population-level response to the rapidly changing selective pressure of phage predation. Results reinforce the importance of phage–host interactions in shaping microbial ecology and evolution over very short time scales.     

Evolution and classification of the CRISPR-Cas systems

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.     

Pooled CRISPR/Cas9 reveals redundant roles of plastidial phosphoglycerate kinases in carbon fixation and metabolism

Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar‐deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis‐dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double‐mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes.