CRISPR/Cas12i3 belongs to the type V-I Cas system, characterized by its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif. Developments of Cas12i3-mediated base editing systems for either C-to-T or A-to-G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3-based cytosine base editor (CBE) only shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as yet. Here, we engineered a series of Cas12i3 (5M)-based CBEs (V0-V5) and ABEs (V0-V5) by fusing a deactivated dCas12i3 (5M) with a transactivation module VP64, a single-stranded DNA-binding domain Rad51, or a double-stranded DNA-binding domain HMG-D, or in combinations, and systemically evaluated their performance in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG-D outperformed other architectures and significantly boosted the efficiencies of Cas12i3 (5M)-based CBE and ABE for C-to-T and A-to-G base editing and expanded the editing window. In stable lines, in comparison to the non-fusion control, the optimized Cas12i3 (5M)-based CBE-V5 and ABE-V5 enabled up to 4.78- and 3.35-fold higher editing efficiencies, with the maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T0 generation. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.

The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif (PAM). However, due to its relatively lower editing efficiency, Cas12i3-mediated multiplex gene editing has not yet been documented in plants. Here, we fused four 5' exonucleases (Exo) including T5E, UL12, PapE, ME15 to the N terminal of an optimized Cas12i3 variant (Cas12i3-5M), respectively, and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines. We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M, respectively. Notably, the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61% across the six tested endogenous genes. We further demonstrated that, although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing, UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three, four, five or six genes with efficiencies of 82.76%, 61.36%, 52.94%, and 51.06% in rice stable lines, respectively. Together, we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice. The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.

Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and its less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3-5M-mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3-5M-mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3-5M increased the gene editing efficiencies by up to 1.34-fold and 3.87-fold, compared to Cas12i3-5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt-T5E-Cas12i3-5M could enhance the editing efficiency by 1.20- to 1.33-fold and 4.05- to 7.95-fold in wheat stable lines compared to Opt-Cas12i3-5M and Opt-Cas12i3, respectively, due to progressive 5'-end resection of the DNA strand at the cleavage site with increased deletion size. The Opt-T5E-Cas12i3-5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt-T5E-Cas12i3-5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement. Here, we isolated a rice endogenous hypoxanthine excision protein, N-methylpurine DNA glycosylase (OsMPG), and engineered two plant A-to-K (K = G or T) base editors, rAKBE01 and rAKBE02, for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor, ABE8e. We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04, respectively. Testing these four rAKBEs, at five endogenous loci in rice protoplasts, indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64. Furthermore, whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus, in comparison with rAKBE02 and rAKBE03, rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57- and 1.75-fold in the rice protoplasts, respectively. Moreover, although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04, rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing, at all the five target loci, with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31% and 1.67 to 4.84% in rice stable lines, respectively. Together, these rAKBEs enable different portfolios of editing products and, thus, now expands the potential of base editing in diverse application scenario for crop improvement.

Background and objectivePost-operative pneumonia (POP), a common complication, may be associated with prolonged hospitalization and long-term mortality in aneurysmal subarachnoid hemorrhage (aSAH) patients. This study aimed to explore the association between pre-operative prognostic nutrition index (PNI) and POP in aSAH patients.MethodsA total of 280 aSAH patients were enrolled from Nanjing Drum Tower Hospital. PNI was calculated as follows: [10 × albumin(gr/dl)] + [0.005 × absolute pre-operative lymphocyte count (per mm3)]. We utilized multivariate analyses, restricted cubic spline, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) to elucidate the role of PNI in POP.ResultsPre-operative PNI levels in the POP group were higher, compared with the non-POP group (41.0 [39.0, 45.4] vs. 44.4 [40.5, 47.3], P = 0.001). When we included PNI as a categorical variable in the multivariate analysis, the levels of PNI were associated with POP (odds ratio, 0.433; 95% confidence interval, 0.253–0.743; P=0.002). In addition, when we included PNI as a continuous variable in the multivariate analysis, the PNI levels were also associated with POP (odds ratio, 0.942; 95% confidence interval, 0.892–0.994; P = 0.028). The level of albumin was also a predictor of the occurrence of POP, with a lower diagnostic power than PNI [AUC: 0.611 (95% confidence interval, 0.549–0.682; P = 0.001) for PNI vs. 0.584 (95% confidence interval, 0.517–0.650; P = 0.017) for albumin]. Multivariable-adjusted spline regression indicated a linear dose–response association between PNI and POP in aSAH participants (P for linearity = 0.027; P for non-linearity = 0.130). Reclassification assessed by IDI and NRI was significantly improved with the addition of PNI to the conventional model of POP in aSAH patients (NRI: 0.322 [0.089–0.555], P = 0.007; IDI: 0.016 [0.001–0.031], P = 0.040).ConclusionThe lower levels of pre-operative PNI may be associated with the higher incidence of POP in aSAH patients. Neurosurgeons are supposed to pay more attention to pre-operative nutrition status in aSAH patients.

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a pre-defined manner by gene editing technologies is highly desirable in crop improvement. Base ed-iting and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates, respectively. Since their discov-eries, various strategies have been attempted to optimize both base editor (BE) and prime editor (PE) in order to improve the precise editing effi-cacy, specificity, and expand the targeting scopes. Here, we summarize the latest develop-ment of various BEs and PEs, as well as their applications in plants. Based on these progresses, we recommend the appropriate BEs and PEs for both basic plant research and crop improvement. Moreover, we propose the perspectives for further optimization of these two editors. We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.

Dear Editor, Base editors(BEs)are promising tools that enable single-nucleo-tide substitutions in specific target genes,generating loss-of-function or gain-of-function mutations,which can greatly accel-erate plant functional genomics and crop improvement(Ren et al.,2018;Zeng et al.,2020;Xu et al.,2021;Yan et al.,2021;Li et al.,2023).To date,three major classes of BEs have been engineered:cytosine BEs(CBEs)for C-to-T transitions(Komor et al.,2016),adenine BEs(ABEs)for A-to-G transitions(Gaudelli et al.,2017),and glycosylase BEs(CGBEs)for C-to-G transversions(Kurt et al.,2021;Zhao et al.,2021).ABE enabling A-to-K(K=G or T)editing remains to be exploited in both mammalian cells and plants.

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore, we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn (D213N) mutation (OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

Dear Editor, Development of a multiplex precision gene editing system is highly desirable for pyramiding beneficial alleles in crop improve-ment.Prime editing is a newly developed genome-editing tool that can precisely enable the installation of all 12 nucleotide sub-stitutions,short insertions,and deletions without exogenous DNA donor repair template and double-strand breaks(Anzalone et al.,2019).Among the prime editing systems,prime editor 3(PE3),which consists of a prime editing guide RNA(pegRNA)and an additional nicking single guide RNA(sgRNA)to nick the non-edited strand,enhances editing efficiency.So far,various strate-gies have been attempted to test the versability of prime editing of a single endogenous gene and to improve its editing efficiency in plants(Li et al.,2020;Tang et al.,2020;Lin et al.,2021;Xu et al.,2021,2022).Multiplex precision gene editing of endogenous genes by prime editing has not been documented in various species,including plants.In this study,we developed three surrogate prime editors,including a hygromycinY46*-based,an OsALSS6271-based,and a combined double surrogate system,for multiplex prime editing of endogenous genes in rice.We then validated the robustness of these three surrogate systems by precision editing of one,two,or three endogenous genes simultaneously,or a combination of precision editing and gene knockouts,in order to expand the applicability of prime editing in simultaneous improvement of multiple traits in crop plants.

Rice blast and bacterial blight, caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively, are devastating diseases affecting rice. Here, we report that a rice valine-glutamine (VQ) motif-containing protein, OsVQ25, balances broad-spectrum disease resistance and plant growth by interacting with a U-Box E3 ligase, OsPUB73, and a transcription factor, OsWRKY53. We show that OsPUB73 positively regulates rice resistance against M. oryzae and Xoo by interacting with and promoting OsVQ25 degradation via the 26S proteasome pathway. Knockout mutants of OsVQ25 exhibit enhanced resistance to both pathogens without a growth penalty. Furthermore, OsVQ25 interacts with and suppresses the transcriptional activity of OsWRKY53, a positive regulator of plant immunity. OsWRKY53 downstream defense-related genes and brassinosteroid signaling genes are upregulated in osvq25 mutants. Our findings reveal a ubiquitin E3 ligase-VQ protein-transcription factor module that fine-tunes plant immunity and growth at the transcriptional and posttranslational levels.

Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.

目的 调查神经外科重症患者合并肺部感染的病原菌分布及耐药情况,并分析其肺部多重耐药菌(MDRO)感染的危险因素.方法 回顾性分析187例伴有肺部感染的神经重症患者的临床资料、肺部病原菌分布和MDRO的耐药情况,并根据其痰培养病原菌是否为多重耐药菌将患者分为耐药菌组和非耐药菌组,分析神经重症伴肺部感染患者感染MDRO的危险因素.结果 肺部非MDRO感染组54例,MDRO感染组133例.共分离出致病菌333株,其中MDRO有196株(58.85％).革兰氏阴性菌(73.98％)为主要致病菌,主要为鲍曼不动杆菌、肺炎克雷伯菌和铜绿假单胞菌.MDRO构成比由高到低依次是鲍曼不动杆菌、金黄色葡萄球菌、肺炎克雷伯菌和铜绿假单胞菌.单因素分析结果显示,入住神经外科重症监护病房(NSICU)时间,最低GCS评分,机械通气时间,人工气道的建立,抗菌药物使用时间及种类,联合用药,碳青霉烯类药物使用和糖皮质激素使用,差异均有统计学意义(均P＜0.05).多因素logistic回归分析结果显示,入住NSICU时间、抗菌药物使用时间及种类是神经重症伴肺部感染患者MDRO感染的危险因素.结论 神经重症患者肺部多重耐药菌感染率高,主要为革兰氏阴性杆菌感染.缩短住院时间,加强抗生素合理使用,减少抗生素使用时间和种类,对控制多重耐药菌感染有重大意义.加速康复外科的实行可能是一项有利措施.

小麦生产关乎国家的粮食安全.品种改良是保持和提高产量的关键.传统育种技术为保障小麦生产做出重要的贡献.小麦需求的不断增加,对育种提出了更高的要求.小麦生物育种技术的快速发展,为进一步提高育种效率提供了新的策略.本文总结了转基因、基因组编辑、基因克隆、分子标记辅助选择和全基因组选择等小麦生物育种技术的最新研究进展,以及生物育种与常规育种、快速育种相结合,在提高小麦育种效率方面的应用.

Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.

目的 观察开颅血肿清除术治疗重型脑干出血的临床效果.方法 重型脑干出血患者12例,格拉斯哥昏迷评分(GCS)3～4分,均采用开颅血肿清除术.术后12 h内复查颅脑CT;术后6个月时,进行格拉斯哥预后量表(GOS)评分.结果 颅脑CT显示,8例血肿接近完全清除(>95％)、4例大部分清除(85％～95％).术后6个月,GOS评分1分5例、2分2例、3分4例、4分1例,其中血肿量≥10 mL者均死亡.结论 开颅血肿清除术治疗重型脑干出血患者临床效果好,但对于血肿量≥10 mL者无效.

Objective:To observe the clinical effect of bevacizumab in the treatment of refractory brain edema.Methods:Eight patients with refractory brain edema who were treated with regular dehydration and hormone for one week in the neurosurgical intensive care unit of Neurosurgery Department of Nanjin Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from July 2020 to June 2021 were selected as the research objects. All patients were given bevacizumab 200 mg, diluted to 0.8 mg/mL with 250 mL normal saline under sterile technology, and continued intravenous drip for more than 90 min. The amount of brain edema and the improvement of GCS score were compared before and after treatment.Results:For patients with refractory brain edema, bevacizumab could significantly improve brain edema. Imaging examination showed that brain edema was improved 2 d after treatment. The amount of brain edema measured 1 week after treatment of all patients were relieved compared with that before treatment, and the GCS score of all patients were higher than that before treatment.Conclusion:Bevacizumab is effective in the treatment of neurosurgical refractory brain edema, which can reduce brain edema and improve the clinical prognosis.