Delivery of CRISPR/Cas9 for therapeutic genome editing

The clustered, regularly‐interspaced, short palindromic repeat (CRISPR)‐associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome‐editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome‐editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off‐target effects. Recently, non‐viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non‐viral vectors, including polymers, liposomes, cell‐penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non‐viral vectors are also discussed.     

An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida)

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-related nuclease 9(Cas9) system enables precise, simple editing of genes in many animals and plants.However, this system has not been applied to rose(Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs(sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspensio...     

植物CRISPR基因组编辑技术的新进展

在过去的4年中,CRISPR/Cas9基因组编辑技术成为生命科学领域的革命性工具,为植物学基础研究和农作物遗传改良提供了高效、快速而又廉价的遗传操作工具。利用CRISPR/Cas9系统可以实现精准的knock-out和knock-in等遗传操作,也可用于靶向激活或抑制基因的表达。在CRISPR/Cas9被广泛地用于基因组编辑的同时,它的编辑能力、效率和精确度也在不断地改进和完善,特别是CRISPR/Cpf1系统的发掘和单碱基编辑技术的创建,使CRISPR系统正逐步成为一个理想的遗传工程技术平台。此外,利用CRISPR/Cas9技术改良的农作物品种也已经涌现,这必将推动精准基因组编辑技术在农作物遗传改良中的应用和发展。     

CRISPR/Cas9在放线菌合成生物学研究中的应用和发展

放线菌是活性天然产物和抗生素药物的重要来源。利用合成生物学高效地开发其中丰富的天然产物资源,将为加速新药开发奠定坚实的基础。CRISPR/Cas9作为一种多功能基因编辑系统,因其便捷高效而被广泛应用于真核生物的遗传操作。但在原核生物尤其是放线菌中的应用仍处于起步阶段,机遇和挑战并存。本综述总结了目前CRISPR/Cas9系统在放线菌基因编辑和调控,以及活性天然产物的产量提升、生物合成机制解析和资源开发等方面的研究进展。同时,也对该系统在应用中面临的包括重组修复效率低,以及靶向切割效率不足等关键挑战进行了分析,并提出了相应的优化解决方法。随着CRISPR/Cas9在放线菌应用中的不断完善和发展,将极大地推动放线菌的合成生物学研究,促进其中天然产物资源的有效挖掘和应用开发。     

Plant genome editing: ever more precise and wide reaching

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.     

基于CRISPR/Cas系统的多重基因编辑与调控技术

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后,凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向,在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善,但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用,针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术,依据作用原理对其进行了系统总结和阐述,包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具,为CRISPR/Cas系统在多领域的应用作出了贡献。     

CRISPR/Cas9技术在工业微生物中的应用

近年来,通过基因编辑技术对工业微生物底盘细胞改造从而获得的优良细胞工厂,促进了农业、医学、环境、能源等领域的可持续发展,提高了人民的生活水平。微生物底盘细胞的改造离不开基因编辑,作为现阶段主要的基因编辑技术,规律间隔成簇短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）/Cas9系统自被发现以来,依靠其低成本、高效率等编辑优点,被广泛用于工业微生物底盘细胞的改造。本文主要简述了以CRISPR/Cas9为基础而衍伸出的各种基因编辑技术,提出了常用的工业微生物对应底盘细胞的改造策略,以期为研究者在进行微生物底盘细胞改造时选择出合适的基因编辑方法。最后指出了CRISPR基因编辑技术面临的PAM位点的依赖性、脱靶效应和应用广泛性等问题。     

CRISPR/Cas9在肿瘤治疗中的研究进展

CRISPR/Cas9技术自从出现以来便迅速应用于肿瘤研究。在肿瘤发生的机理研究中,CRISPR/Cas9可用于研究单核苷酸突变、染色体异位等因素在肿瘤发生中的作用机制,同时也可以用于肿瘤细胞中功能缺陷基因的筛选。在肿瘤治疗方法的研究中,CRISPR/Cas9主要用于诱发机制比较清晰且诱因为病毒的肿瘤类型,例如鼻咽癌、宫颈癌等,通过对相应病毒的基因进行编辑从而抑制其致癌作用。利用CRISPR/Cas9技术还可以加速新肿瘤治疗靶点基因的发现。尽管发展和应用十分迅速,但是CRISPR/Cas9在肿瘤研究和治疗中的作用仍然受多种因素的限制,包括Cas9和sgRNA的输送效率、脱靶效应以及安全性和成本等。对CRISPR/Cas9在肿瘤研究中的应用进展进行了综述,以期为肿瘤发生、转移机制和肿瘤治疗等方面的研究提供参考。     

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.     

CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

Expression of PTEN‐long mediated by CRISPR/Cas9 can repress U87 cell proliferation

PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN‐long is an alternative translation variant, with an additional 173 amino acids added to the N‐terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN‐long is secreted into serum and can re‐enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells. As an alternative approach, if a therapeutic protein can be directly delivered to target cell of interest, it should theoretically function well within the cells, particularly for genes that are deficiently expressed in vivo. Most therapeutic proteins are incapable of efficiently permeating the cell membrane. In this study, we have employed CRISPR/Cas9 gene editing tool combined with single‐stranded template to edit CTG of PTEN‐long to ATG in the genome. Two guide RNAs close to CTG site were found to have similar efficiency in driving PTEN‐long expression. Furthermore, we detected PTEN‐long expression in transfected whole‐cell lysate and in concentrated culture media in Western blot. Interestingly, the culture media of PTEN‐long expression can reduce Akt phosphorylation level and repress U87 cell proliferation compared to wild‐type U87 or control media. Taken together, PTEN‐long driven by CRISPR/Cas9 imports and exports cells and represses nearby cell proliferation, indicating the PTEN‐long generated by CRISPR/Cas9 has potential to be an alternative strategy for PTEN gene therapy.     

CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展

CRISPR/Cas9基因组编辑技术是一项对基因组进行精准修饰的技术,可实现对靶标基因的碱基插入、缺失或DNA片段替换。随着人们对CRISPR/Cas9系统的了解逐渐加深,其在科研、农业和医疗等领域的应用也越来越广泛。该文简要介绍了CRISPR/Cas9基因组编辑技术的发展以及工作原理,总结了近几年对该技术进行优化与改进的研究进展,包括基因组编辑效率的提升、基因组编辑范围的扩展、单碱基精准编辑以及多基因同时编辑、基因组编辑安全性的提升以及基因片段替换与基因靶向转录调控,以期为深入开展这一领域的研究提供参考。     

利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展

CRISPR/Cas9基因组编辑技术是一项对基因组进行精准修饰的技术, 可实现对靶标基因的碱基插入、缺失或DNA片段替换。随着人们对CRISPR/Cas9系统的了解逐渐加深, 其在科研、农业和医疗等领域的应用也越来越广泛。该文简要介绍了CRISPR/Cas9基因组编辑技术的发展以及工作原理, 总结了近几年对该技术进行优化与改进的研究进展, 包括基因组编辑效率的提升、基因组编辑范围的扩展、单碱基精准编辑以及多基因同时编辑、基因组编辑安全性的提升以及基因片段替换与基因靶向转录调控, 以期为深入开展这一领域的研究提供参考。     

CRISPR/Cas9基因组编辑技术在HIV-1感染治疗中的应用进展

基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的。因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-1原病毒患者的治疗中具有重大的现实意义。目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题。最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), CRISPR/Cas9]已被成功改造成基因组定点编辑工具。因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点。本文参考最新国内外研究成果,重点介绍了 CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考。     

Recent Advances in CRISPR‐Cas9 Genome Editing Technology for Biological and Biomedical Investigations

The Type II CRISPR‐Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single‐mutation genetic diseases, and multiple‐site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR‐Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81–94, 2018. © 2017 Wiley Periodicals, Inc.