Anti‐HIV‐1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication

The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.     

CRISPR-derived genome editing technologies for metabolic engineering

In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.     

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.     

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm,Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA(gRNA), they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy.We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines,one expressing Cas9 protein in the posterior silk glands(PSGs) and the other constitutively expressing BmlaminA/C(BmLMN) gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%,and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.     

Simple Genome Editing of Rodent Intact Embryos by Electroporation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.     

基于CRISPR-Cas系统的基因组定点修饰新技术

CRISPR(clustered regulatory interspersed short palindromic repeat)序列源于原核生物的一种获得性免疫系统,协同Cas(CRISPR-associated)蛋白家族参与抵抗噬菌体或其它病毒的二次感染,广泛存在于细菌(60%)和古菌(90%)中.病菌和宿主的共同进化导致了CRISPR-Cas系统具有多样性,可分为3大类(Ⅰ-Ⅲ),又分为10亚类.在Ⅱ型CRISPR-Cas系统基础上建立了RNA介导的CRISPR-Cas系统来修饰(删除、添加、激活、抑制)靶细胞中特定的基因序列,现已在人类细胞、小鼠、斑马鱼、酵母、细菌、果蝇、线虫、拟南芥中得以应用.本文主要介绍了Ⅱ型CRISPR-Cas系统的结构特点、作用机理及作为新型基因组定点修饰技术的研究进展,分析该技术优势,并展望CRISPRCas系统的应用前景.     

Data Comparison and Software Design for Easy Selection and Application of CRISPR-based Genome Editing Systems in Plants

CRISPR-based genome editing systems have been successfully and effectively used in many organisms. However, only a few studies have reported the comparison between CRISPR/Cas9 and CRISPR/Cpf1 systems in the whole-genome applications. Although many web-based toolkits are available, there is still a shortage of comprehensive, user-friendly, and plant-specific CRISPR databases and desktop software. In this study, we identified and analyzed the similarities and differences between CRISPR/Cas9 and CRISPR/Cpf1 systems by considering the abundance of proto-spacer adjacent motif (PAM) sites, the effects of GC content, optimal proto-spacer length, potential universality within the plant kingdom, PAM-rich region (PARR) inhibiting ratio, and the effects of G-quadruplex (G-Q) structures. Using this information, we built a comprehensive CRISPR database (including 138 plant genome data sources, www.grapeworld.cn/pc/index.html), which provides search tools for the identification of CRISPR editing sites in both CRISPR/Cas9 and CRISPR/Cpf1 systems. We also developed a desktop software on the basis of the Perl/Tk tool, which facilitates and improves the detection and analysis of CRISPR editing sites at the whole-genome level on Linux and/or Windows platform. Therefore, this study provides helpful data and software for easy selection and application of CRISPR-based genome editing systems in plants.     

嗜热厌氧杆菌热稳定CRISPR/Cas9基因组编辑技术及在细胞工厂构建中的应用研究进展

嗜热厌氧杆菌属(Thermoanaerobacter)来源的菌株作为一个高温发酵的细胞工厂,具有生产生物燃料和化学品的潜力,已引起了研究者的广泛兴趣.随着嗜热厌氧杆菌属来源的多个菌株全基因组测序的完成以及相关的生理生化实验的开展,建立一个简便、快捷的基因操作技术将有助于进一步深入理解和认识嗜热厌氧杆菌有关的代谢途径.最...     

CRISPR-OTE: Prediction of CRISPR On-Target Efficiency Based on Multi-Dimensional Feature Fusion

ObjectiveClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a powerful genome editing technology. Guide RNA (gRNA) plays an essential guiding role in the CRISPR system by complementary base pairing with target DNA. Since the CRISPR targeting mechanism problem has not yet been fully resolved, it remains a challenge to predict gRNA on-target efficiency. Current gRNA design tools often lack efficient information extraction and cannot learn the target efficiency patterns thoroughly.Material and methodsIn this study, CRISPR-OTE is proposed to consider both multi-dimensional sequence information and important complementary prior knowledge based on a simple but effective framework. CRISPR-OTE consists of the local-contextual information branch and the prior knowledge branch. The local-contextual information branch extracts multi-dimensional sequence features from the DNA primary sequence by a parallel framework of Convolutional Neural Networks (CNN) and bidirectional Long Short-Term Memory networks (biLSTM). The prior knowledge branch selects the optimal subset of physicochemical features to provide the neural network with complementary knowledge, such as complex secondary structures. A simple feature fusion strategy is also adopted to fully utilize multi-modal data from the two branches.ResultsThe experimental results show that the optimal subset of physicochemical features (RNA secondary structure and melting temperature of 34nt target) can effectively improve the prediction performance. Additionally, combining multi-dimensional sequence features and multi-modal features can extract information more comprehensively. Through transfer learning, CRISPR-OTE trained on the CRISPR-Cpf1 system can also be successfully applied to the CRISPR-Cas9 system.ConclusionThe performance of CRISPR-OTE is superior to other methods in different CRISPR systems and species. Therefore, CRISPR-OTE is a simple on-target efficiency prediction framework with better accuracy and generalization performance.     

谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势.目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱...     

Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.     

Diversity of CRISPR-Cas-mediated mechanisms of adaptive immunity in prokaryotes and their application in biotechnology

CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.     

Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations

Plant Molecular Biology - We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for...     

Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm

Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.     

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。     

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.     

GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana

The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9) is simple and highly efficient technology applied to functional studies of genes and genetic crop improvement. In this study, we have demonstrated the utility of green fluorescent protein (GFP) marker to detect the targeting efficiency of gRNAs. As a proof of concept, Glycine max De-Etiolated 1 (GmDET1) gene was chosen and tagged with GFP to rapidly analyze genome editing efficiency of gRNAs. Results showed weaker GFP fluorescence signal in the N. benthamiana leaves co-infiltrated with GmDET1-GFP overexpression (OE)?+?DET1 gRNA1 constructs as compared to the stronger GFP florescence signal in the leaves co-infiltrated with DET1 gRNA2 and gRNA3 constructs, thus indicating the highest of DET1 gRNA1. These results were further confirmed by the detection of the mutation frequencies through T7 endonuclease (T7E1) assay and sequencing; the highest mutation rate of 38.46% in GmDET1 targeted by DET1 gRNA1 to that of DET1 gRNA2 (7.69%) and gRNA3 (15.38%) was observed. Thus our studies showed “GFP tagging” as the most reliable and rapid method-one can apply to minimize the generation of non-edited transgenic plants resulting from inefficient gRNAs.