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Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives.
CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector's expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.
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Protocol for targeted modification of the rice genome using base editing.
Base editing is a precision genome-editing approach that is widely utilized to generate single-nucleotide variants (SNVs) in genomes. Here, we present a protocol to perform targeted adenine (A)-to-guanine (G) substitution in rice using adenine base editor (ABE). We detail the design of sgRNA, CRISPR plasmid construction, rapid genetic transformation of rice, and genotyping of editing events. This protocol can be applied to cytosine base editing in rice as well. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021).1.
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Gene and Base Editing as a Therapeutic Option for Cystic Fibrosis-Learning from Other Diseases.
Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some animal studies have clearly established that precision genome editing can be significantly more effective than cDNA addition. These observations have led to a number of gene-editing clinical trials for a small number of such genetic disorders. To date, gene-editing strategies to correct CFTR mutations have been conducted exclusively in cell models, with no in vivo gene-editing studies yet described. Here, we highlight some of the key breakthroughs in in vivo and ex vivo gene and base editing in animal models for other diseases and discuss what might be learned from these studies in the development of editing strategies that may be applied to cystic fibrosis as a potential therapeutic approach. There are many hurdles that need to be overcome, including the in vivo delivery of editing machinery or successful engraftment of ex vivo-edited cells, as well as minimising potential off-target effects. However, a successful proof-of-concept study for gene or base editing in one or more of the available CF animal models could pave the way towards a long-term therapeutic strategy for this disease.
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Base Editing Landscape Extends to Perform Transversion Mutation.
Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.
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BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.
Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.
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Genetic Manipulation of MRSA Using CRISPR/Cas9 Technology.
The clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system has emerged as an efficient genome engineering method attributed to its high efficiency and versatility. By generating a lethal double-strand DNA break in the target genome, the CRISPR/Cas9 system is capable of selecting the separated crossover events occurring in the traditional genetic manipulation methods in one step, therefore enabling rapid and efficient genome editing in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). By engineering the fusion of a cytidine deaminase APOBEC1 and a Cas9 nickase, a base editor was further developed as a highly efficient gene inactivation and point mutation tool in S. aureus. Here we describe a detailed protocol for CRISPR/Cas9-based genome editing in S. aureus, including genome modification and base editing. This protocol outlines in detail the design of primers, the construction and transformation of editing plasmids, as well as the verification of sequence-specific CRISPR/Cas9-mediated mutagenesis in S. aureus.
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Efficient Genome and Base Editing in Human Cells Using ThermoCas9.
Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.
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CRISPR/Cas9 Editing to Facilitate and Expand Drug Discovery.
INTRODUCTION: The ability of most laboratories to easily access CRISPR/Cas9 engineering tools has caused a revolution in biology. One of the areas that will continue to be impacted by genome editing is the drug discovery process. OBJECTIVE: CRISPR/Cas9 will not only serve to accelerate the drug discovery pipeline, but also streamline line it by identifying high-value targets, facilitating the validation of drug: target interactions and mechanisms of action, and stimulating the development of phenotype-based high throughput screens as alternatives to target-based assays. CONCLUSION: We review the literature and hurdles that have been overcome to develop the current generation of tools being used to enrich the drug discovery paradigm.
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Cytosine base editing systems with minimized off-target effect and molecular size.
Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector.
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A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing.
Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future.
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