A revolutionary tool: CRISPR technology plays an important role in construction of intelligentized gene circuits

With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence‐specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR‐associated RNA‐guided endonuclease Cas9 (CRISPR‐associated protein 9)‐targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties‐operability, high efficiency and programmability. The traditional single‐targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti‐tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour‐specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch‐inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR‐based gene circuit development.     

CRISPR/Cas9技术在感染性疾病中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other 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 Editing in Biological and Biomedical Investigation

The CRISPR (clustered regularly interspaced short palindromic repeat)‐Cas (CRISPR‐associated protein) system, a prokaryotic RNA‐based adaptive immune system against viral infection, is emerging as a powerful genome editing tool in broad research areas. To further improve and expand its functionality, various CRISPR delivery strategies have been tested and optimized, and key CRISPR system components such as Cas protein have been engineered with different purposes. Benefiting from more in‐depth understanding and further development of CRISPR, versatile CRISPR‐based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine. In biological research area, CRISPR has been widely adopted in both fundamental and applied research fields, such as genomic and epigenomic modification, genome‐wide screening, cell and animal research, agriculture transforming, livestock breeding, food manufacture, industrial biotechnology, and gene drives in disease agents control. In biomedical research area, CRISPR has also shown its extensive applicability in the establishment of animal models for genetic disorders, generation of tissue donors, implementation of antimicrobial and antiviral studies, identification and assessment of new drugs, and even treatment for clinical diseases. However, there are still several problems to consider, and the biggest concerns are the off‐target effects and ethical issues of this technology. In this prospect article, after highlighting recent development of CRISPR systems, we outline different applications and current limitations of CRISPR in biological and biomedical investigation. Finally, we provide a perspective on future development and potential risks of this multifunctional technology. J. Cell. Biochem. 119: 52–61, 2018. © 2017 Wiley Periodicals, Inc.     

一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA （double-stranded DNA,dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system,DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair,HDR）效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。     

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.     

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.     

Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1

Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR‐Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off‐target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR‐Cas12b systems for genome editing in Arabidopsis.     

基于CRISPR/Cas9技术的基因敲入/敲除策略

成簇的规律间隔性短回文序列(CRISPR)基因编辑系统,因其设计简单操作方便和无种属限制,已成为一种广泛应用的基因组定点编辑工具,在复杂的基因组编辑,例如基因的人源化改造以及条件等位基因的构建中有所应用。在自然界中,CRISPR系统拥有多种类别。其中,CRISPR/Cas9系统是研究最深入、应用最成熟的一种。本文针对CRISPR/Cas9系统,分别从基因敲入/敲除片段的大小、同源臂长短、构型即递送方式等技术环节进行综述,阐述不同设计及操作条件下由CRISPR/Cas9系统介导的基因敲入/敲除的效率差异。     

Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

β‐thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2‐654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β‐thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double‐strand break (DSB), which can be combined with the single‐stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2‐654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β‐Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.     

CRISPR-Cas系统在植物中的研究进展与监管政策

基因编辑技术作为一种颠覆性新技术,现已广泛应用于作物的遗传改良,显示出巨大的发展潜力和应用价值。各国在加快技术研发的同时也十分关注其可能带来的安全性问题,相继出台了基因编辑作物的安全监管政策。综述了目前常用的CRISPR基因编辑系统的原理, 最新开发的一系列CRISPR变体,CRISPR系统在植物中的应用,基因编辑植物检测方法及国际上的相关监管政策,以期为我国基因编辑作物监管政策的制定提供理论数据。     

Extensive mapping of an innate immune network with CRISPR

The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al ( 2015 ) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs. Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.