中国科学家发现胞嘧啶单碱基编辑工具存在 基因组范围的脱靶

基于CRISPR-Cas的单碱基编辑工具是近2年基因组编辑技术的重大突破之一, 已经在人类(Homo sapiens)细胞和动植物中得到了验证与应用。最近, 中国科学家分析了胞嘧啶编辑器(CBE) BE3和HF1-BE3, 以及腺嘌呤编辑器(ABE)等单碱基编辑工具在水稻(Oryza sativa)中的脱靶现象, 发现BE3和HF1-BE3两个CBE在全基因组范围内存在脱靶编辑, 而ABE则没有脱靶现象。这一发现对单碱基编辑工具的应用和进一步改进具有重要意义。     

中国科学家发现胞嘧啶单碱基编辑工具存在 基因组范围的脱靶

基于CRISPR-Cas的单碱基编辑工具是近2年基因组编辑技术的重大突破之一, 已经在人类(Homo sapiens)细胞和动植物中得到了验证与应用。最近, 中国科学家分析了胞嘧啶编辑器(CBE) BE3和HF1-BE3, 以及腺嘌呤编辑器(ABE)等单碱基编辑工具在水稻(Oryza sativa)中的脱靶现象, 发现BE3和HF1-BE3两个CBE在全基因组范围内存在脱靶编辑, 而ABE则没有脱靶现象。这一发现对单碱基编辑工具的应用和进一步改进具有重要意义。     

Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing

Base editing has emerged as a revolutionary technology for single nucleotide modifications. The cytosine and adenine base editors (CBEs and ABEs) have demonstrated great potential in clinical and fundamental research. However, screening and isolating target-edited cells remains challenging. In the current study, we developed a universal Adenine and Cytosine Base-Editing Antibiotic Resistance Screening Reporter (ACBE-ARSR) for improving the editing efficiency. To develop the reporter, the CBE-ARSR was first constructed and shown to be capable of enriching cells for those that had undergone CBE editing activity. Then, the ACBE-ARSR was constructed and was further validated in the editing assays by four different CBEs and two versions of ABE at several different genomic loci. Our results demonstrated that ACBE-ARSR, compared to the reporter of transfection (RoT) screening strategy, improved the editing efficiency of CBE and ABE by 4.6- and 1.9-fold on average, respectively. We found the highest CBE and ABE editing efficiencies as enriched by ACBE-ARSR reached 90% and 88.7%. Moreover, we also demonstrated ACBE-ARSR could be employed for enhancing simultaneous multiplexed genome editing. In conclusion, both CBE and ABE activity can be improved significantly using our novel ACBE-ARSR screening strategy, which we believe will facilitate the development of base editors and their application in biomedical and fundamental research studies.     

小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立

目的:利用单碱基编辑技术定点修饰MECP2基因T158M位点,获得单一定点突变类型的啮齿类及非人灵长类胚胎,为建立模拟临床上Rett综合征的疾病动物模型奠定基础。方法:利用单碱基编辑技术构建3对T158M-sgRNA质粒,并分别与BE系统(BE3或BE4max)质粒共转染293T细胞筛选高效的sgRNA,通过显微注射将体外转录的mRNA以不同的浓度组合注射到ICR小鼠和猕猴受精卵中,检测小鼠和猕猴胚胎发育率和编辑效率,评估BE3和BE4max的工作效率及最优浓度组合。结果:小鼠胚胎上BE4max(ng/μl)∶sgRNA(ng/μl)=100∶50浓度组合时,小鼠胚胎发育率和编辑效率最佳,同时该浓度组合在猕猴胚胎上也获得了有效编辑效率。结论:成功在小鼠及猕猴胚胎上实现了MECP2基因T158M位点上C→T的转变,为后期建立T158M突变的RTT动物模型建立了稳定的胚胎编辑体系。     

BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发及应用

碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶(rAPOBEC1)与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低(0-20%);在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口(PAM上游-11到-19位),有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。     

Amide-based derivatives of β-alanine hydroxamic acid as histone deacetylase inhibitors: Attenuation of potency through resonance effects

A library of amide-linked derivatives of β-alanine hydroxamic acid were prepared (2-7) and the activity as inhibitors of Zn(II)-containing histone deacetylases (HDACs) determined in vitro against HDAC1 and the anti-proliferative activity determined in BE(2)-C neuroblastoma cells. The IC(50) values of the best-performing compounds (3-7) against HDAC1 ranged between 38 and 84μM. The least potent compound (2) inhibited a maximum of only 40% HDAC1 activity at 250μM. The anti-proliferative activity of 2-7 at 50μM against BE(2)-C neuroblastoma cells ranged between 57.0% and 88.6%. The structural similarity between the potent HDAC inhibitor trichostatin A (TSA, 1; HDAC1, IC(50) 12nM) and the present compounds (2-7) was high at the Zn(II) coordinating hydroxamic acid head group; and in selected compounds (2, 5), at the 4-(dimethylamino)phenyl tail. The significantly reduced potency of 2-7 relative to 1 underscores the rank importance of the linker region as part of the HDAC inhibitor pharmacophore. Molecular modeling of 1-7 using HDAC8 as the template suggested that the conformationally constrained 4'-methyl group of 1 may contribute to HDAC inhibitor potency through a sandwich-like interaction with a hydrophobic region containing F152 and F208; and that the absence of this group in 2-7 may reduce potency. The close proximity of the 5'-carbonyl oxygen atom in 2-7 to the sulfur atom of Met274 in HDAC8 or the corresponding isobutyl group of Leu274 in HDAC1 may attenuate potency through repulsive steric and dipole-dipole forces. In a unique resonance stabilized form of 2, this interaction could manifest as stronger ion-dipole repulsive forces, resulting in a further decrease in potency. This work suggests that resonance structures of HDAC inhibitors could modulate intermolecular interactions with HDAC targets, and potency.     

Precise A•T to G•C base editing in the zebrafish genome

Background

Base editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. Recently, adenine base editors (ABEs) have been developed to mediate the conversion of A?T to G?C in genomic DNA of human cells, mice, and plants. Here, we investigated the activity and efficiency of several adenine base editors in zebrafish and showed that base editing can be used to create new models of pathogenic diseases caused by point mutations.

Results

The original ABE7.10 exhibits almost no activity in zebrafish. After codon optimization, we found that a zABE7.10 variant could induce targeted conversion of adenine to guanine in zebrafish at multiple tested genomic loci, and all the target sites showed a high rate of germline targeting efficiency. Furthermore, using this system, we established a zebrafish model of 5q-Syndrome that contained a new point mutation in rps14. The further modification of zABE7.10 by a bipartite nuclear localization signals (bpNLS) resulted in 1.96-fold average improvement in ABE-mediated editing efficiency at four sites.

Conclusions

Collectively, this system, designated as zABE7.10, provides a strategy to perform A?T to G?C base editing in zebrafish and enhances its capacity to model human diseases.

     

毕赤酵母胞嘧啶碱基编辑器的设计与功能评价

【目的】在巴斯德毕赤酵母（Pichia pastoris）中建立一套分子靶向突变系统，为毕赤酵母的基因工程改造提供高效的编辑工具。【方法】基于规律成簇的间隔短回文重复序列/Cas9核酸酶（clustered regularly interspaced short palindromic repeats/Cas9 nuclease，CRISPR/Cas9）技术，设计并构建nCas9与胞苷脱氨酶融合表达的胞嘧啶碱基编辑器（cytosine base editor，CBE），并选择酵母基因组中富含碱基C的一段序列作为靶标以评价CBE的碱基编辑功能。电转化酵母后，利用高通量测序技术分析CBE的编辑效率及编辑模式，并进一步探究连接肽长度、融合蛋白相对位置和gRNA靶向序列（即spacer）长度等因素对CBE功能的影响。【结果】nCas9与PmCDA1融合组成的CBE能够实现毕赤酵母基因组碱基C的高效编辑。当连接肽长度为（GGGGS）₁₀时，CBE的编辑效率最高，编辑窗口位于前间隔序列邻近基序（protospacer adjacent motif，PAM）远端的C20–C14之间，其中C18的编辑效率可达85.1%。nCas9与PmCDA1相对位置的改变对CBE的编辑效率和编辑模式的影响不大。而gRNA靶向序列长度影响着CBE的编辑效率，且gRNA靶向序列长度不能低于17 nt，但19–23 nt之间均可引导CBE对基因组的高效编辑。【结论】本研究在巴斯德毕赤酵母中构建了一套具有高效碱基编辑活性的胞嘧啶碱基编辑器，为基于毕赤酵母的基础和应用研究提供了工具支持。     

Base editing‐mediated perturbation of endogenous PKM1/2 splicing facilitates isoform‐specific functional analysis in vitro and in vivo

Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet.Materials and methodsIn this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development.ResultsThe splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms.ConclusionsThis work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.     

Structural and energetic basis for the inhibitory selectivity of both catalytic domains of dimeric HDAC6

Abstract

HDAC6 is a protein involved in cancer, neurodegenerative disease and inflammatory disorders. To date, the full three-dimensional (3D) structure of human HDAC6 has not been elucidated; however, there are some experimental 3D structural homologs to HDAC6 that can be used as templates. In this work, we utilized molecular modeling procedures to model both of the catalytic domains of HDAC6 connected by the linker region where DMB region is placed. Once the 3D structure of human HDAC6 was obtained, it was structurally evaluated and submitted to docking and molecular dynamic (MD) simulations along with Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method to explore the stability and the binding free energy properties of the HDAC6–ligand complexes. In addition, its structural and energetic behavior was explored with each one of the catalytic domains in the molecular recognition of six selective HDAC6 inhibitors, HPOB, CAY10603, Nexturastat, Rocilinostat, Tubacin and Tubastatin A for DD2, and with the so-called 9-peptide which is DD1–HDAC6 selective substrate. The use of the whole system (DD1–DMB–DD2) showed a tendency toward the ligand affinity of DD2, CAY10603> Tubacin?>?Rocilinostat?> Nexturastat?>?HPOB?>?Tubastatin > 9-peptide, which is in line with experimental reports. However, 9-peptide showed a higher affinity for DD1, which agrees with experimental reports elsewhere. Principal component analysis provided important information about the structural changes linked to the molecular recognition process, whereas per-residue decomposition analysis revealed the energetic contribution of the key residues in the molecular binding and structural characteristics that could assist in drug design.     

Design,synthesis and activity evaluation of indole-based double – Branched HDAC1 inhibitors

Histone deacetylases inhibitors (HDACIs) represents effective treatments for cancer. In continuing our efforts to develop novel and potent HDACIs, a series of N-hydroxycinnamamide-based HDACIs with aromatic ring and various aliphatic linker have been successfully designed and synthesized. Biological evaluations established that compounds 4h, 4i, 4j, 4l, 4r showed superior inhibition on histone deacetylase and antiproliferative activity in some solid tumor cell lines [HeLa, SK-N-BE(2), PC-3] compared to the known inhibitor SAHA. Among these analogs, 4l exhibited selectivity to HDAC1.     

Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis

Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red–like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp^D10A) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.     

Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1, N11-bis(ethyl)norspermine

Properties of a mutant form of spermidine/spermine N(1)-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N(1,) N(11)-bis(ethyl)norspermine (BE 3-3-3) were investigated. Increased K(m) values, decreased V(max) values, and decreased k(cat) values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also approximately 20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced approximately 300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.     

BEguider:一个单碱基编辑器sgRNA设计与编辑效率预测工具

单碱基编辑器是实用且高效的基因编辑工具,其编辑效率与单向导RNA(single guide RNA, sgRNA)序列的设计密切相关。目前单碱基编辑器sgRNA序列的设计缺少特定的法则,主要依靠经验和大量尝试完成。本研究基于卷积神经网络,开发了一个单碱基编辑器sgRNA序列设计工具BEguider。BEguider利用TensorFlow 2深度学习框架建立编辑效率预测模型,能够在人基因组范围内针对NGG PAM序列依赖的单碱基编辑器ABE7.10-NGG和BE4-NGG批量设计sgRNA序列,预测编辑效率。此外,通过整合Cas-OFFinder, BEguider能够提供对sgRNA脱靶情况的评估。利用BEguider设计sgRNA序列,有助于研究人员提高实验效率,节约实验成本。