Versatile Genome Editing Tool Research Articles

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, invivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Addition of the T5 exonuclease increases the prime editing efficiency in plants

Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5′-to-3′ exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dual-pegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.

Predicting prime editing efficiency and product purity by deep learning.

Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications.

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

BackgroundCRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. ResultsA total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. ConclusionThis technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

Genome editing in maize: Toward improving complex traits in a global crop

Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.

High‐efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR‐Cas systems

AbstractThermus thermophilus is an attractive species in the bioindustry due to its valuable natural products, abundant thermophilic enzymes, and promising fermentation capacities. However, efficient and versatile genome editing tools are not available for this species. In this study, we developed an efficient genome editing tool for T. thermophilus HB27 based on its endogenous type I‐B, I‐C, and III‐A/B CRISPR‐Cas systems. First, we systematically characterized the DNA interference capabilities of the different types of the native CRISPR‐Cas systems in T. thermophilus HB27. We found that genomic manipulations such as gene deletion, mutation, and in situ tagging could be easily implemented by a series of genome‐editing plasmids carrying an artificial self‐targeting mini‐CRISPR and a donor DNA responsible for the recombinant recovery. We also compared the genome editing efficiency of different CRISPR‐Cas systems and the editing plasmids with donor DNAs of different lengths. Additionally, we developed a reporter gene system for T. thermophilus based on a heat‐stable β‐galactosidase gene TTP0042, and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.

General guidelines for CRISPR/Cas-based genome editing in plants.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.

DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.

The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A-/-, Dnmt3b-/-) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings.

Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range

The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Pairing of single mutations yields obligate Cre-type site-specific recombinases.

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.

Versatile Applications of the CRISPR/Cas Toolkit in Plant Pathology and Disease Management

New tools and advanced technologies have played key roles in facilitating basic research in plant pathology and practical approaches for disease management and crop health. Recently, the CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR-associated) system has emerged as a powerful and versatile tool for genome editing and other molecular applications. This review aims to introduce and highlight the CRISPR/Cas toolkit and its current and future impact on plant pathology and disease management. We will cover the rapidly expanding horizon of various CRISPR/Cas applications in the basic study of plant-pathogen interactions, genome engineering of plant disease resistance, and molecular diagnosis of diverse pathogens. Using the citrus greening disease as an example, various CRISPR/Cas-enabled strategies are presented to precisely edit the host genome for disease resistance, to rapidly detect the pathogen for disease management, and to potentially use gene drive for insect population control. At the cutting edge of nucleic acid manipulation and detection, the CRISPR/Cas toolkit will accelerate plant breeding and reshape crop production and disease management as we face the challenges of 21st century agriculture.

Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review.

The emergence of genome-editing technology has allowed manipulation of DNA sequences in genomes to precisely remove or replace specific sequences in organisms resulting in targeted mutations. In plants, genome editing is an attractive method to alter gene functions to generate improved crop varieties. Genome editing is thought to be simple to use and has a lower risk of off-target effects compared to classical mutation breeding. Furthermore, genome-editing technology tools can also be applied directly to crops that contain complex genomes and/or are not easily bred using traditional methods. Currently, highly versatile genome-editing tools for precise and predictable editing of almost any locus in the plant genome make it possible to extend the range of application, including functional genomics research and molecular crop breeding. Vegetables are essential nutrient sources for humans and provide vitamins, minerals, and fiber to diets, thereby contributing to human health. In this review, we provide an overview of the brief history of genome-editing technologies and the components of genome-editing tool boxes, and illustrate basic modes of operation in representative systems. We describe the current and potential practical application of genome editing for the development of improved nutritious vegetables and present several case studies demonstrating the potential of the technology. Finally, we highlight future directions and challenges in applying genome-editing systems to vegetable crops for research and product development.

Genome editing with the CRISPR-Cas system: an art, ethics and global regulatory perspective.

SummaryOver the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products.

CRISPR-Cas genome editing tool: a narrow lane of cancer therapeutics with potential blockades.

In recent, clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas) system is emerging as a versatile genome editing tool with applications in basic science, preclinical and translational biology. This CRISPR-Cas genome editing tool is known as a precise and effective option to correct a part of the genome that may have implications in many human diseases including cancer associated genes such as oncogenes and onco-suppressors. Besides robust potential to edit target genes, CRISPR-Cas editing technology displays cellular alterations in the form of activation of DNA double strand break repair system and bringing genomic instability. As a consequence of repair of DNA double strand breaks, highly mitotically active cells may face hyper-DNA repair systems and there may be sometimes a situation leading to error prone mutations and unwanted genomic integrity. Additionally, the use of CRISPR-Cas editing technology in cancer therapy is limited in the backdrop of genotype and epigenomic heterogeneity in tumors. Therefore, a precaution should be considered to employ CRISPR-Cas technology in cancer therapy in view of tumor heterogeneity and environmental pressure.

MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing - a robust and cost-effective method to genotype multiple targets in a single reaction. Our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.

RNAi-mediated control of CRISPR functions.

CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material.Methods: In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs.Results: amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both in vitro and in vivo and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs.Conclusion: Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.

Era of Genomic Medicine: A Narrative Review on CRISPR Technology as a Potential Therapeutic Tool for Human Diseases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) provides acquired immunity in microorganisms against exogenous DNA that may hinder the survival of the organism. Pioneering work by Doudna and Charpentier in 2012 resulted in the creation of the CRISPR/Cas9 genome editing tool on the basis of this concept. The aim of this was to create a rapid, efficient, and versatile genome-editing tool to facilitate genetic manipulation. The mechanism relies on two components: the RNA guide which acts as a sentinel and a Cas protein complex which functions as a highly precise molecular knife. The guide RNA can be modified to match a DNA sequence of interest in the cell and accordingly be used to rectify mutations that may otherwise cause disease. Within a few years following the development of the CRISPR/Cas9 tool, its usage has become ubiquitous. Its influence extends into many fields of biological sciences from biotechnology and biochemistry to molecular biology and biomedical sciences. The following review aims at shedding some light on to the applications of the CRISPR/Cas9 tool in the field of biomedical sciences, particularly gene therapy. An insight with relation to a few of the many diseases that are being tackled with the aid of the CRISPR/Cas9 mechanism and the trends, successes, and challenges of this application as a gene therapy are discussed in this review.

Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.

Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.