Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence

Rapeseed (Brassica napus),an important oilseed crop,has adapted to diverse climate zones and latitudes by forming three main ecotype groups,namely winter,semiwinter,and spring types. However,genetic variations underlying the divergence of these ecotypes are largely unknown. Here,we report the global pattern of genetic polymorphisms in rapeseed determined by resequencing a worldwide collection of 991 germplasm accessions.A total of 5.56 and 5.53 million singlenucleotide polymorphisms (SNPs)as Well as 1.86 and 1.92 million InDels were identified by mapping reads to the reference genomes of "Darmor-bzh"and "Tapidor,"respectively.We generated a map of allelic drift paths that shows splits and mixtures of the main populations,and revealed an asymmetric evolution of the two subgenomes of B.napus by calculating the genetic diversity and linkage disequilibrium parameters.Selective-sweep analysis revealed genetic changes in genes orthologous to those regulating various aspects of plant development and response to stresses.A genome-wide association study identified SNPs in the promoter regions of FLOWERING LOCUS T and FLOWERING LOCUS C orthologs that corresponded to the different rapeseed ecotype groups. Our study provides important insights into the genomic footprints of rapeseed evolution and flowering-time divergence among three ecotype groups,and will facilitate screening of molecular markers for accelerating rapeseed breeding.

Gene editing and its applications in biomedicine

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors

Programmed cell death protein-1(PD-1)-mediated immunosuppression has been proposed to contribute to the limited clinical efficacy of chimeric antigen receptor T(CAR-T)cells in solid tumors.We generated PD-1 and T cell receptor(TCR)deficient mesothelin-specific CAR-T(MPTK-CAR-T)cells using CRISPR-Cas9 technology and evaluated them in a dose-escalation study.A total of 15 patients received one or more infusions of MPTK-CAR-T cells without prior lymphodepletion.No dose-limiting toxicity or unexpected adverse events were observed in any of the 15 patients.The best overall response was stable disease(2/15 patients).Circulating MPTK-CAR-T cells peaked at days 7–14 and became undetectable beyond 1 month.TCR-positive CAR-T cells rather than TCR-negative CAR-T cells were predominantly detected in effusion or peripheral blood from three patients after infusion.We further confirmed the reduced persistence of TCR-deficient CAR-T cells in animal models.Our results establish the preliminary feasibility and safety of CRISPR-engineered CAR-T cells with PD-1 disruption and suggest that the natural TCR plays an important role in the persistence of CAR-T cells when treating solid tumors.

CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

T cells engineered with chimeric antigen receptor （CAR） have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CART efficacy. One negative regulator of T cell activity is lymphocyte activation gene-3 （LAG-3）. We successfully generated LAG-3 knockout T and CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CART cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia

Myeloid-derived suppressor cells(MDSCs)are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity.Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity.Our previous study demonstrated that high-dose dexamethasone(HD-DXM)corrected the functional impairment of MDSCs in immune thrombocytopenia(ITP);however,the MDSC population was not restored in nonresponders,and the mechanism remained unclear.In this study,altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders,accompanied by decreased levels of carnitine palmitoyltransferase-1(CPT-1),a rate-limiting enzyme in fatty acid oxidation(FAO).Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders.We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor(GR),which can translocate into mitochondria to regulate the transcription of mitochondrial DNA(mtDNA)as well as the level of oxidative phosphorylation.It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs.Finally,by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs,we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP.In conclusion,our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

In vitro transcribed sgRNA causes cell death by inducing interferon release

Dear editor The clustered regularly in terspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins 9 (Cas9) systems are powerful tools for gene editing. Ribonucleoprotein (RNP) complex composed of Cas9 pro? tein and sgRNA binds to specific genomic loci and gen erate DNA double strand breaks. While plasmids expressing Cas9 protein and sgRNA are routinely transfected into various cell lines to perform gene editing (Cong et al., 2013;Mali et al., 2013;Ran et al., 2013), direct delivery of Cas9-sgRNA RNP has shown higher efficiency and lower off-target effects (Kim et al., 2014), especially in human primary cells such as T cells (Hendel et al., 2015;Schumann et al., 2015). SgRNA can be gen erated by either in vitro transcript! on (IVT) or chemical syn thesis. IVT is widely used to gen erate sgRNAs, since it can be easily performed in most labs.

Ng Ago: an exciting new tool for genome editing

Site-directed DNA endonucleases are powerful tools for genome editing.When introduced into cells,these systems can bind to a target DNA sequence in the genome and create a DNA double-strand break(DSB),the repair

Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation

Previously we established Zygote Electroporation of Nucleases（ZEN） technology as an efficient and high-throughput way to generate genetically modified mouse models.However,there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol.In this study,we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation.Using this approach,we were able to introduce precise nucleotide substitutions,large segment deletion and short segment insertion into targeted loci with high efficiency.

5'capped and 3'polyA-tailed sgRNAs enhance the efficiency of CRISPR-Cas9 system

Dear Editor,The clustered regularly interspaced short palindromic repeats(CRISPR)-associated(Cas)system is an adaptive immune system in a variety of bacteria and archaea(Terns and Terns,2011).The most commonly used Streptococcus pyogenes type II CRISPR-Cas9 system consists of Cas9 nuclease and two short RNAs,crRNA and tracrRNA,which can be linked together forming one chimeric single guide RNA(sgRNA)(Jinek et al.,2012).

Gene editing in T cell therapy

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for further improvements of T cell therapy. Here we review the applications of gene editing in various T cell therapies, focusing on antiviral strategies and cancer immunotherapies, and discuss the challenges and future prospects.

Recent advances in CRISPR research

The clustered regularly interspaced short palindromic repeats(CRISPR)technology has revolutionized life sciences and developed rapidly.Here,we highlight the recent advances in development and application of CRISPR technologies,including the discovery of novel CRISPR systems,CRISPR base editing and imaging,and the applications of CRISPR in plant breeding,animal breeding,disease modeling and biotherapy.

Temperature effect on CRISPR-Cas9 mediated genome editing

Zinc-finger nuclease（ZFN）, transcription activator-like effector nuclease（TALEN）, and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9（CRISPR-Cas9） are the most commonly used genome editing tools. Previous studies demonstrated that hypothermia treatment increased the mutation rates induced by ZFNs and TALENs in mammalian cells. Here, we characterize the effect of different culture temperatures on CRISPR-Cas9 mediated genome editing and find that the genome editing efficiency of CRISPR-Cas9 is significantly hampered by hypothermia treatment, unlike ZFN and TALEN. In addition, hyperthermia culture condition enhances genome editing by CRISPR-Cas9 in some cell lines, due to the higher enzyme activity and sg RNA expression level at higher temperature. Our study has implications on CRISPR-Cas9 applications in a broad spectrum of species, many of which do not live at 37 C.

Questions about NgAgo

Dear Editor： Gao et al. published data in Nature Biotechnology （Nat Biotechnol. 2016 May 2） showing that DNA-guided genome editing using the Natronobacterium gregoryi Argonaute （NgAgo） protein targeted 47 mammalian genomic loci with a 100% success rate and an efficiency of 21.3%-41.3% at various targets. This report led us to test NgAgo＇s utility in various cells and organisms such as mouse and zebrafish for gene editing.

Erratum to： Questions about NgAgo

In the original publication of this article the Figure 1E legend has been incorrectly published.

Extended pluripotent stem cells facilitate mouse model generation

Mouse embryonic stem(mES)cells,established in 1981(Evans and Kaufman,1981;Martin,1981),were derived from the inner cell mass(ICM)of blastocysts and can be expanded in vitro for many passages,maintaining normal karyotype and differentiation potential.Upon introduction into blastocysts,mES cells can differentiate into all three germ layers,contributing to all the somatic lineages and germline.

A brief review of genome editing technology for generating animal models

The recent development of genome editing technologies has given researchers unprecedented power to alter DNA sequences at chosen genomic loci,thereby generating various genetically edited animal models.This mini-review briefly summarizes the development of major genome editing tools,focusing on the application of these tools to generate animal models in multiple species.

Editing base in mouse model

Genome editing technologies enable scientists to modify DNA sequence at specific genomic loci in various cells and species. There are several editing tools, including homing endonucleases （Silva et al., 2011）, zinc finger nucleases （ZFN） （Urnov et al., 2010）, transcription activator-like effector nucleases （TALENs） （Bogdanove and Voytas, 2011）, and clustered regularly interspaced short palindromic repeats （CRISPR） and the CRISPR-associated proteins （Cas） system （Doudna and CharDentier, 2014）.

Genome editing in plants using the TnpB transposase system

The widely used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas)system is thought to have evolved from IS200/IS605 transposons.TnpB proteins,encoded by one type of IS200/IS605 transposon,are considered to be the evolutionary ancestors of Cas12 nucleases,which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells.TnpB nucleases,which are smaller than Cas nucleases,have been engineered for use in genome editing in animal systems,but the feasibility of this approach in plants remained unknown.Here,we obtained stably transformed genome-edited mutants in rice(Oryza sativa)by adapting three recently identified TnpB genome editing vectors,encoding distinct TnpB nucleases(ISAam1,ISDra2,and ISYmu1),for use in plants,demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes.ISDra2 and ISYmu1 precisely edited their target sequences,with no off-target mutations detected,showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants.Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs.