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.     

CRISPR/Cas9-mediated correction of human genetic disease

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas) protein 9 system(CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs,this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity,and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining(NHEJ) and homology directed repair(HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.     

基于CRISPR/Cas9的激活系统研究进展

基因编辑技术自问世以来就一直作为生物技术领域的研究热点。基因编辑工具成簇的规律间隔短回文重复序列及其相关系统(CRISPR/Cas系统)具有特异性、简便性和灵活性等优点,为研究人员提供了丰富的遗传操作工具,也让CRISPR/Cas系统的应用在多种生物中得到了飞速发展。特别是将转录激活因子与失活的Cas蛋白结合,可在RNA转录水平实现基因表达特异性调控,为生物技术在医学研究及农业领域的发展做出了重要的贡献。外源基因的过表达是验证基因功能和基因调控的常用方法,然而由于载体容量的限制难以实现多基因过表达。基于CRISPR/Cas9激活系统可在不同向导RNA的引导下对多个基因进行调控,实现调控水平验证基因功能。本文通过对CRISPR/Cas9激活系统组成及不同激活策略进行总结,整理针对过度激活的解决方案,为CRISPR/Cas9激活系统应用于棉花遗传改良及除草剂抗性研究提供更多参考。     

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off‐target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR‐induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5‐fold in somatic tissues and up to 100‐fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double‐stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on‐target mutagenesis in plants using CRISPR/Cas9.     

A justice‐based argument for including sickle cell disease in CRISPR/Cas9 clinical research

CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health‐care system, and benefit‐sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene

The CRISPR/Cas technology has been successfully used to stimulate the integration of small DNA sequences in a target locus to produce gene mutations. However, many applications require homologous recombination using large gene-targeting constructs. Here we address the potential of CRISPR/Cas-mediated double-strand breaks to enhance the genetic engineering of large target sequences using a construct for “humanizing” the mouse Cnr2 gene locus. We designed a small-guide RNA that directs the induction of double strand breaks by Cas9 in the Cnr2 coding exon. By co-transfection of the CRISPR/Cas system with the 10 kb targeting construct we were able to boost the recombination frequency more than 200-fold from 0.27% to 67%. This simple technology can thus be used for the homologous integration of large gene fragments and should greatly enhance our ability to generate any kind of genetically altered mouse models.     

机器学习方法在CRISPR/Cas9系统中的应用

基于CRISPR/Cas9系统介导的第三代基因组定点编辑技术,已被广泛应用于基因编辑和基因表达调控等研究领域。如何提高该技术对基因组编辑的效率与特异性、最大限度降低脱靶风险一直是该领域的难点。近年来,机器学习为解决CRISPR/Cas9系统所面临的问题提供了新思路,基于机器学习的CRISPR/Cas9系统已逐渐成为研究热点。本文阐述了CRISPR/Cas9的作用机理,总结了现阶段该技术面临的基因组编辑效率低、存在潜在的脱靶效应、前间区序列邻近基序(PAM)限制识别序列等问题,最后对机器学习应用于优化设计高效向导RNA (sgRNA)序列、预测sgRNA的活性、脱靶效应评估、基因敲除、高通量功能基因筛选等领域的研究现状与发展前景进行了展望,以期为基因组编辑领域的研究提供参考。     

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。     

A Mouse Geneticist’s Practical Guide to CRISPR Applications

CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos.     

CRISPR/Cas9系统在基因组DNA片段编辑中的应用

源于细菌和古菌的Ⅱ型成簇规律间隔短回文重复系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9),CRISPR/Cas9]近年被改造成为基因组定点编辑的新技术。由于它具有设计简单、操作方便、费用低廉等巨大优势,给遗传操作领域带来了一场革命性的改变。本文重点介绍了CRISPR/Cas9系统在基因组DNA片段靶向编辑方面的研究和应用,主要包括DNA片段的删除、反转、重复、插入和易位,这一有效的DNA片段编辑方法为研究基因功能、调控元件、组织发育和疾病发生发展提供了有力手段。本文最后展望了Ⅱ型CRISPR/Cas9系统的应用前景和其他类型CRISPR系统的应用潜力,为开展利用基因组DNA片段靶向编辑进行基因调控和功能研究提供参考。     

Key elements for designing and performing a CRISPR/Cas9-based genetic screen

Reverse genetic screens are invaluable for uncovering gene functions, but are traditionally hampered by some technical limitations. Over the past few years, since the advent of the revolutionary CRISPR/Cas9 technology, its power in genome editing has been harnessed to overcome the traditional limitations in reverse genetic screens, with successes in various biological contexts. Here, we outline these CRISPR/Cas9-based screens, provide guidance on the design of effective screens and discuss the potential future directions of development of this field.     

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction(PCR)/restriction enzyme(RE) assay, T7 endonuclease I(T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting(HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR(ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.     

CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.     

诺贝尔化学奖授予CRISPR-Cas9基因编辑研究

2020年，诺贝尔化学奖授予现在德国马普感染生物学研究所工作的法国科学家Emmanuelle Charpentier和美国加州大学伯克利分校的?Jennifer Doudna，表彰她们发明CRISPR基因编辑方法。她们揭示了Cas9具有RNA介导的DNA 核酸内切酶活性，可以切断任意DNA双链，产生DNA双链断裂。她们还指出CRISPR具有在活细胞中修改基因的能力，利用CRISPR-Cas9编辑工具人们可以精确改变细胞中的DNA。由于简单、高效、廉价等特征，CRISPR已经成为全球最为流行的基因编辑技术，被称为编辑基因的“魔剪”。本文介绍两位诺贝尔化学奖获得者的研究成果，总结CRISPR系统的发现过程，并概述CRISPR-Cas9的功能以及应用。     

Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing

The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.