Efficient large fragment deletion in plants:double pairs of sgRNAs are better than dual sgRNAs

Dear Editor,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a powerful and versatile gene editing system that has been extensively utilized in various animals and plants,which holds enormous potential and value for scientific research and breeding.However,single-targeted CRISPR can only induce a few base deletions,insertions,or substitution.Ideally,thesemutations result in premature termination of the protein encoded by the target gene,leading to a loss of function[1].

聚碳酸酯光学制品注塑成型的残余应力

采用单一变量实验考察了不同注射工艺参数对光学制品双折射的分布规律,通过应力-光弹定律计算了对称轴上残余应力值。用Moldflow软件对制品成型过程中型腔内流场进行模拟,分析了厚度方向应力的变化。结果表明,沿熔体流动方向上,制品残余应力是逐渐减小的,浇口附近有应力集中;在合理工艺范围内,适当提高成型温度和模具温度,延长保压时间和冷却时间有助于减小制品的残余应力,保压压力和注塑速度应适中才能改善制品的成型质量。

超声波表征天然橡胶/环氧化天然橡胶并用体系的硫化过程

在料腔安装超声波探头的无转子硫化仪上同步监测天然橡胶(NR)/环氧化天然橡胶(ENR)硫化过程得到了声速曲线和扭矩曲线,对比其相似性提出了超声波表征硫化历程的方法,指出其对微观结构和密实度变化更加灵敏。在压力-体积-温度(PVT)-超声波检测装置上同步测试了该并用胶的比容和声速,这两种同步测试数据可用直线方程、考虑压力效应的声阻抗方程和从Tait方程推导直接反映温度效应的方程对硫化过程进行较好描述,说明超声波还可直接监测硫化胶的比容变化。

交联聚乙烯回收料填充高密度聚乙烯的发泡特性

采用机械粉碎法制备得到的交联聚乙烯回收料(RXLPE)填充高密度聚乙烯(HDPE)以提高其熔体强度,并用偶氮二甲酰胺(AC)作为发泡剂在烘箱实现了无压发泡。结果表明,RXLPE加入HDPE后界面相容,部分解交联RXLPE微团分散在HDPE中形成微观"网络"结构,减小了HDPE熔体流动性,但提高了熔体强度和拉伸黏度及"应变硬化"效应。加入发泡助剂氧化锌(ZnO)和硬脂酸锌(ZnSt2)时,AC分解温度从205℃降低到160℃左右,可在低温发泡形成更均匀、粒径更小的泡孔。低于30%含量,增大RXLPE用量可制备发泡率高和强度大的发泡品;RXLPE/HDPE为30∶70时,发泡倍率最高可达6倍多。RXLPE/HDPE同HDPE发泡体受压呈现典型的小变形线弹性、中变形塌陷和大变形密实化的三区行为;适量添加RXLPE和发泡助剂可改善后两区性能;过多RXLPE导致泡孔壁曲折而大变形时响应小。

Cas9蛋白变体VQR高效识别水稻NGAC前间区序列邻近基序

成簇的规律间隔短回文重复序列及CRISPR相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated9,CRISPR/Cas9)系统是近年来发展起来并被广泛应用的第三代基因组编辑工具。但是,该系统的酿脓链球菌Cas9(Streptococcus pyogenes, SpCas9)仅能识别NGG前间区序列邻近基序(protospacer adjacent motif, PAM),极大地限制了基因组编辑的范围。SpCas9变体VQR(D1135V/R1335Q/T1337R)在水稻中可识别NGAA、NGAG和NGAT PAM,但尚不清楚是否能识别NGAC PAM。本研究利用改进后的CRISPR/VQR系统对水稻中3个相对低效的VQR靶位点NAL1-Q1、NAL1-Q2和LPA1-Q进行了编辑,结果表明改进后的CRISPR/VQR系统可以高效编辑这3个靶位点,编辑效率分别为9.75%、43.90%和29.26%。为了明确改进后的CRISPR/VQR系统对NGAC PAM的识别情况,本研究选择水稻叶片宽度调控基因NARROW LEAF 1 (NAL1)中的NAL-C位点和蜡质合成基因GLOSSY1(GL1)中的GL1-C位点进行基因编辑,并获得57株转基因水稻。靶位点PCR扩增及测序结果表明,NAL1-C和GL1-C靶标位点突变的植株分别为27株和44株,突变率分别为47.36%和77.19%;其中NAL1-C/GL1-C双突变植株为26株,双突变率为45.61%。进一步分析表明,CRISPR/VQR系统造成的突变有4种类型,分别为杂合突变、双等位突变、嵌合体突变和纯合突变,其中以杂合突变和双等位突变为主。这些结果表明,改进的CRISPR/VQR系统可以高效编辑水稻NGAC PAM位点,并产生丰富的突变类型。本研究为水稻及其他植物相关基因NGAC PAM位点的编辑提供了理论依据。

Cellular apoptosis and Caspase-12 expression in a rat model of progressive spinal cord compression

BACKGROUND： Studies have demonstrated that the mechanisms underlying cellular apoptosis signal transduction focus on two pathways： intracellular mitochondria and extracellular death receptor. The current evidence supports that signal transduction of cellular apoptosis also includes endoplasmic reticulum stress signal transduction. OBJECTIVE： To observe Caspase-12 expression and cellular apoptosis following ischemia in rats with progressive spinal cord compression, and to verify the influence of endoplasmic reticulum stress on the apoptosis induced by spinal cord injury. DESIGN, TIME AND SETTING： A randomized, controlled, animal trial was performed at the Institute of Neuroscience in Chongqing Medical University between January and October in 2006. MATERIALS： Immunohistochemical kit, diaminobenzidine, and TUNEL kit were purchased from Beijing Zhongshan Biotechnology, China; rabbit anti-rat Caspase-12 monoclonal antibody was provided by Santa Cruz, USA. METHODS： Sixty Wistar rats, aged 3-4 months, were randomly assigned to a model group （n = 50）, which underwent spinal cord compression in the L1 segment following L1 laminectomy and articular process excision to establish a model of progressive spinal cord compression, and a sham-surgery group （n = 10）, which underwent only laminectomy. Starting with the first day after surgery, the rats were locally anesthetized, the skin was opened, and the screw was rotated by 1/4 of a cycle, twice weekly. MAIN OUTCOME MEASURES： At 3, 7, 14, 21, and 28 days after surgery, rats from each group were anesthetized, and the spinal cords were resected. Pathological changes following spinal cord compression were determined using hematoxylin-eosin staining, Nissl dye, and transmission electron microscopy. The TUNEL method was used to observe neuronal apoptosis in the compressed spinal cord segments. Immunohistochemistry and Western blot were utilized to detect Caspase-12 expression in the compressed segments. RESULTS： Cellular swelling, neural degeneration, and altered endoplasmic reticulum structures were observed at 3 days following compression. Symptoms became gradually aggravated with increasing compression time. Compared with the sham-surgery group, the number of apoptotic neurons was remarkably increased in compressed segments of the model group （P 〈 0.05）, and Caspase-12 expression was also shown to increase （P 〈 0.05）. CONCLUSION： Neuronal apoptosis was a predominant pathological factor resulting in secondary spinal cord injury during progressive spinal cord compression, and Caspase-12 was shown to be possibly involved in neuronal apoptosis induced by progressive spinal cord compression.

CRISPR/Cas systems: The link between functional genes and genetic improvement

With the ever-increasing human population and deteriorating environmental conditions,there is an urgent need to breed environmentally friendly and resource-conserving rice cultivars to achieve sustainable agricultural development and food security.However,conventional rice improvement strategies,such as hybrid breeding,are time-consuming and laborious processes and may not be able to keep pace with increasing food demand in the future.Targeted genome-editing technologies,especially clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein(CRISPR/Cas),permit efficient targeted genome modification and offer great promise for the creation of desired plants with higher yield,improved grain quality,and resistance to herbicides,diseases,and insect pests.There is also great potential for tapping heterosis using the CRISPR/Cas technology.In this review,we focus on the most essential applications of CRISPR/Cas genome editing to rice genetic improvement,considering traits such as yield,quality,and herbicide,disease and insect-pest resistance.We discuss applications of CRISPR/Cas to the exploitation of heterosis.Finally,we outline perspectives for future rice breeding using genome-editing technologies.

Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.

Coexistence of dopamine-beta-hydroxylase and activated protein-2 alpha in rat cerebellar Purkinje cells

BACKGROUND： Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum. Numerous reports have also been published addressing whether dopamine-beta-hydroxylase （DBH） expression exists in cerebellar Purkinje cells. OBJECTIVE： To investigate the coexistence of DBH and activator protein-2α expression in rat cerebellar Purkinje cells. DESIGN, TIME AND SETTING： A cell morphological study was performed at the Institute of Neuroscience, Chongqing Medical University, China in May 2007. MATERIALS： Ten healthy Wistar rats, of either gender, aged 14 weeks, served as experimental animals. Rabbit anti-mouse DBH, goat anti-mouse activator protein-2α and rabbit anti-mouse β-actin （Santa Cruz Biotechnology, Inc., USA）, horseradish peroxidase-labeled goat anti-rabbit IgG, FITC-labeled mouse anti-rabbit IgG, and Cy3-labeled mouse anti-goat IgG （Boster, Wuhan, China）, were used in this study. METHODS： Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α, with double-label immunofluorescence being employed to determine coexpression of both, in the cerebellum of 5 randomly selected rats. Western blot assay was utilized to determine the expression of DBH and activator protein-2α in the cerebellum of the remaining 5 rats. MAIN OUTCOME MEASURES： Expression, localization and coexistence of DBH and activator protein-2α in the cerebellum were measured separately. RESULTS： Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α. Western blot assay also demonstrated DBH and activator protein-2α expression in the cerebellum. Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2α in cerebellar Purkinje cells. CONCLUSION： Norepinephrine and activator protein-2α coexist in rat cerebellar Purkinje cells.

Synthetic apomixis with normal hybrid rice seed production

Heterosis has long been exploited in the hybrid seed industry,which contributes to high and stable yields of modern agriculture(Huang et al.,2016).However,heterosis phenotypes of hybrid plants are segregated in its offspring.Apomixis allows instant fixation and propagation though seeds with heterozygous genotypes,showing great potential in plant breeding and agricultural practice(Ye and Cui,2019).Apomixis naturally occurs in hundreds of plant species,but it is absent in major crop species(Underwood and Mercier,2022).Recently,synthetic apomixis has been engineered in rice by combining Mitosis instead of Meiosis(MiMe)with a mutation of MATRILINEAL or ectopic expression of BABY BOOM1(BBM1),enabling clonal reproduction of F1 hybrids through seeds and stable transmission of heterotic phenotypes over generations(Khanday et al.,2019;Wang et al.,2019;Liu et al.,2022).However,the fertility of both two strategies was significantly reduced compared with that of the wild type,which hinders the application of both strategies in agriculture.In this study,we established synthetic apomixis with a high fertility that is almost comparable to normal hybrid rice.

Manipulation of genetic recombination by editing the transcriptional regulatory regions of a meiotic gene in hybrid rice

Dear Editor,Genetic breeding involves the recombination and selection of various valuable genes.Meiotic crossover(CO)promotes the generation of new allelic combinations on chromosomes,which is essential for breeding elite varieties(Wijnker and de Jong,2008).An increase in CO promotes genetic diversity,whereas a decrease can rapidly stabilize excellent traits(Mercier et al.,2015).Furthermore,the complete elimination of CO facilitates heterotic fixation during apomixis(Wang et al.,2019).

Characterizing 86-attosecond isolated pulses based on amplitude gating of high harmonic generation[Invited]

Isolated attosecond pulses(IAPs)are generated via applying amplitude gating on high-order harmonic generation driven by carrier-envelope phase stabilized 5.2 fs pulses with 0.5 mJ pulse energy at 770 nm central wavelength at the Synergetic Extreme Condition User Facility.A continuum ranging from 70 to 100 eV that supports sub-100-attosecond pulse is extracted by Zr foil and Mo/Si multilayer mirror.We demonstrate the characterization of the IAP.The retrieved pulse duration is 86attoseconds.The developed attosecond laser beamline with repetition rate up to 10 kHz is available for users to conduct attosecond photoelectron spectroscopy researches with a capability of coincidence measurement.

Liquiritigenin promotes osteogenic differentiation and prevents bone loss via inducing auto-lysosomal degradation and inhibiting apoptosis

Osteoporosis(OP)is a debilitating skeletal abnormality involving bone remodeling and bone cell homeostasis characterized by decreased bone strength and high fracture risk.A novel therapeutic intervention for OP by manipulating cellular autophagy-apoptosis processes to promote skeletal homeostasis is presented.Protective effects of the naturally occurring plant extract Liquiritigenin(LG)were demonstrated in an ovariectomy(OvX)-OP mouse model and preosteoblast MC3T3-E1 cells.Micro-CT and histological staining assessments of skeletal phenotype were applied alongside detection of autophagy activity in osteocytes and MC3T3-E1 cells by transmission electron microscopy(TEM).The effects of LG on chloroquine(CQ)-and the apoptosis-inducing TS-treated osteogenic differentiations and status of lysosomes within MC3T3-E1 cells were analyzed by Neutral red,Alizarin red S and alkaline phosphatase(ALP)staining and Western blot assays.Treatment with LG prevented bone loss,increased osteogenic differentiation in vivo and in vitro,and inhibited osteoclast formation to some extent.TEM analyses revealed that LG can improve auto-lysosomal degradation within osteocytes from OVX mice and MC3T3-E1 cells.The abnormal status of lysosomes associated with CQ and TS treatments was notably alleviated by LG which also reduced levels of apoptosis-induced inhibition of osteogenic differentiation and averted abnormal osteogenic differentiation as a consequence of a blockage in autolysosome degradation.Overall,LG stimulates bone growth in Oovx mice through increased osteogenic differentiation and regulation of autophagyapoptosis mechanisms,presenting an auspicious natural therapy for Op.

Synthetic apomixis enables stable transgenerational transmission of heterotic phenotypes in hybrid rice

In hybrid plants,heterosis often produces large,vigorous plants with high yields;however,hybrid seeds are generated by costly and laborious crosses of inbred parents.Apomixis,in which a plant produces a clone of itself via asexual reproduction through seeds,may produce another revolution in plant biology.Recently,synthetic apomixis enabled clonal reproduction of F_(1) hybrids through seeds in rice(Oryza sativa),but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear.Here,we propagated clonal plants to the T_(4) generation and investigated their genetic and molecular stability at each generation.By analyzing agronomic traits,as well as the genome,methylome,transcriptome,and allele-specific transcriptome,we showed that the descendant clonal plants remained stable.Unexpectedly,in addition to normal clonal seeds,the plants also produced a few aneuploids that had eliminated large genomic segments in each generation.Despite the identification of rare aneuploids,the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems

The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.

A Simple CRISPR/Cas9 System for Multiplex Genome Editing in Rice

Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.

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.

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

Dear Editor,The CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/Cas9)system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs(PAMs).To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs

Developing disease-resistant thermosensitive male sterile rice by multiplex gene editing

High-quality and disease-resistant male sterile lines have great potential for applications in hybrid rice breeding.We introduced specific mutations into the TMS5,Pi21,and Xa13 genes in Pinzhan intermediate breeding material using the CRISPR/Cas9 multiplex genome editing system.We found that the transgenefree homozygous triple tms5/pi21/xa13 mutants obtained in the T1 generation displayed characteristics of thermosensitive genic male sterility(TGMS)with enhancedresistance to rice blast and bacterial blight.Our study provides a convenient and effective way of converting breeding intermediate material into TGMS lines through multiplex gene editing,which could significantly accelerate the breeding of sterile lines.

Targeted mutagenesis in rice using CRISPR-Cpf1 system

Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.