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。     

Small-molecule compounds boost genome-editing efficiency of cytosine base editor

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.     

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.     

Expanding the base editing scope in rice by using Cas9 variants

Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology‐directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.     

基于CRISPR/Cas系统的DNA碱基编辑技术及其在生物医学和农业中的应用

近年来,基于成簇的规律间隔短回文重复序列及其相关系统(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas)的基因编辑技术飞速发展,该系统可以利用同源定向重组(Homology directed repair,HDR)来完成其介导的精准编辑,但效率极低,限制了其在农业和生物医学等领域上的推广应用。基于CRISPR/Cas系统的DNA碱基编辑技术作为一种新兴的基因组编辑技术,能在不产生双链断裂的情况下实现碱基的定向突变,相对于CRISPR/Cas介导的HDR编辑具有更高的编辑效率和特异性。目前,已开发出了可将C碱基突变为T碱基的胞嘧啶碱基编辑器(Cytidine base editors,CBE),将A碱基突变为G碱基的腺嘌呤碱基编辑器(Adenine base editors,ABE),以及可实现碱基任意变换和小片段精准插入和缺失的Prime编辑器(Prime editors,PE)。另外,能实现C到G颠换的糖基化酶碱基编辑器(Glycosylase base editors,GBE)以及能同时编辑A和C两种底物的双碱基编辑器也已被开发出来。文中主要综述了几种DNA碱基编辑器的开发历程、研究进展及各自优点和局限性;介绍了DNA碱基编辑技术在生物医学以及农业中的成功应用案例,以期为DNA碱基编辑器的进一步优化和选择应用提供借鉴。     

碱基编辑器的开发及其在细菌基因组编辑中的应用

碱基编辑器是近两年发展起来的新型基因组编辑工具,它将碱基脱氨酶的催化活性和CRISPR/Cas系统的靶向特异性进行结合,催化DNA或RNA链上特定位点的碱基发生脱氨基反应,进而完成碱基的替换。碱基编辑器分为DNA和RNA碱基编辑器两大类,其中DNA碱基编辑器分为两种:胞嘧啶碱基编辑器和腺嘌呤碱基编辑器;前者可以实现胞嘧啶到胸腺嘧啶的转换,而后者则可以将腺嘌呤突变为鸟嘌呤。由于DNA碱基编辑器不会造成DNA的双链断裂(DSB),也不依赖于宿主的非同源末端修复和同源重组途径,因此,大大减少了DSB相关的编辑副产物,如小片段插入或缺失等。基于CRISPR/Cas系统的RNA碱基编辑器,可以实现RNA链上腺嘌呤核苷到次黄苷的转换。本文对不同类型碱基编辑器的开发过程、适用范围和编辑特点等进行梳理,并对其在细菌基因组编辑中的应用进行了介绍;最后简要探讨了细菌中碱基编辑器的缺点以及将来可能的研究方向。     

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。     

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。     

Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing

CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.     

碱基编辑系统研究最新进展及应用

CRISPR系统能够在基因组DNA中完成精准编辑,但依赖于细胞内的同源重组(Homologydirected recombination,HDR)修复途径,且效率极低。基于CRISPR/Cas9系统开发的碱基编辑技术(Base editing)通过将失去切割活性的核酸酶与不同碱基脱氨基酶融合,构建了两套碱基编辑系统(Baseeditors,BE):胞嘧啶碱基编辑器(Cytosine base editor,CBE)和腺嘌呤碱基编辑器(Adenine base editor,ABE)。这两类编辑器分别能够在不产生DNA双链断裂的前提下在基因靶位点完成CT (GA)或AG (TC)的替换,最终实现精准的碱基编辑。目前碱基编辑技术已经广泛应用于基因治疗、动物模型构建、精准动物育种和基因功能分析等领域,为基础和应用研究提供了强大的技术工具。文中概括了碱基编辑技术的研发过程、技术优势、应用现状、存在问题及改进策略,以期为相关领域的科研人员了解和使用碱基编辑系统提供参考。     

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.

     

Simplified adenine base editors improve adenine base editing efficiency in rice

Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE‐P1S (Adenine Base Editor‐Plant version 1 Simplified) containing the ecTadA*7.10‐nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE‐P1 consisting of the ecTadA‐ecTadA*7.10‐nSpCas9 (D10A) fusion. We found that the protein expression level of ABE‐P1S is higher than that of ABE‐P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE‐P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10‐nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH‐Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.     

AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.     

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.     

A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants

CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.     

基因编辑之“新宠”—单碱基基因组编辑系统

近年发展起来的人工核酸酶可通过引起特定位点的DNA双链断裂实现对目的片段的有效编辑。为进一步提高碱基修改的效率和精确度,2016年研究者们利用CRISPR/Cas9识别特定DNA序列的功能,结合胞嘧啶脱氨酶的生化活性发明了将胞嘧啶高效转换为胸腺嘧啶(C>T)的嘧啶单碱基编辑系统(base editor)。这一系统虽然能精准实现嘧啶直接转换,大大提高精确基因编辑效率,但美中不足的是无法对嘌呤进行修改。近期,Nature报道了将细菌中的tRNA腺嘌呤脱氨酶定向进化形成具有催化DNA腺嘌呤底物的脱氨酶,将其与Cas9系统融合发明了具有高效催化腺嘌呤转换为鸟嘌呤的新工具—腺嘌呤单碱基编辑系统(ABEs, adenine base editors)。本文总结了单碱基编辑工具的发展历程和最新研究进展,着重介绍ABEs的研发过程,并对单碱基编辑工具今后的应用方向和研发方向进行展望。