Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus

Lactobacillus, a genus of lactic acid bacteria, plays a crucial function in food production preservation, and probiotics. It is particularly important to develop new Lactobacillus strains with superior performance by gene editing. Currently, the identification of its functional genes and the mining of excellent functional genes mainly rely on the traditional gene homologous recombination technology. CRISPR/Cas9-based genome editing is a rapidly developing technology in recent years. It has been widely applied in mammalian cells, plants, yeast, and other eukaryotes, but less in prokaryotes, especially Lactobacillus. Compared with the traditional strain improvement methods, CRISPR/Cas9-based genome editing can greatly improve the accuracy of Lactobacillus target sites and achieve traceless genome modification. The strains obtained by this technology may even be more efficient than the traditional random mutation methods. This review examines the application and current issues of CRISPR/Cas9-based genome editing in Lactobacillus, as well as the development trend of CRISPR/Cas9-based genome editing in Lactobacillus. In addition, the fundamental mechanisms of CRISPR/Cas9-based genome editing are also presented and summarized.     

Assembly of the Complete Mitochondrial Genome of Gelsemium elegans Revealed the Existence of Homologous Conformations Generated by a Repeat Mediated Recombination

Gelsemium elegans (G. elegans) is a Chinese medicinal plant with substantial economic and feeding values. There is a lack of detailed studies on the mitochondrial genome of G. elegans. In this study, the mitochondrial genome of G. elegans was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of G. elegans is represented by two circular chromosomes of 406,009 bp in length with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected 145 pairs of repeats and found that four pairs of repeats could mediate the homologous recombination into one major conformation and five minor conformations, and the presence of conformations was verified by PCR amplification and Sanger sequencing. A total of 124 SSRs were identified in the G. elegans mitochondrial genome. The homologous segments between the chloroplast and mitochondrial genomes accounted for 5.85% of the mitochondrial genome. We also predicted 477 RNA potential editing sites and found that the nad4 gene was edited 38 times, which was the most frequent occurrence. Taken together, the mitochondrial genome of G. elegans was assembled and annotated. We gained a more comprehensive understanding on the genome of this medicinal plant, which is vital for its effective utilization and genetic improvement, especially for cytoplasmic male sterility breeding and evolution analysis in G. elegans.     

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T₀ plants and 83.2% of T₁ plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T₁ progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.     

Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing

The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100–200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.     

Intracellular Delivery of mRNA for Cell-Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles

Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.     

A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50–52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.     

Virus-Induced Gene Editing and Its Applications in Plants

CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.     

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.     

Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00–13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.     

Enhancing Animal Disease Resistance,Production Efficiency,and Welfare through Precise Genome Editing

The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation have been fundamentally revolutionized. Agricultural animals with traits of interest can be obtained in just one generation (and without long time selection). Here, we reviewed the advancements of the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) genome editing tools and their applications in animal breeding, especially in improving disease resistance, production performance, and animal welfare. Additionally, we covered the regulations on genome-edited animals (GEAs) and ways to accelerate their use. Recommendations for how to produce GEAs were also discussed. Despite the current challenges, we believe that genome editing breeding and GEAs will be available in the near future.     

Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.     

Exosomes as Targeted Delivery Platform of CRISPR/Cas9 for Therapeutic Genome Editing

Therapeutic genome editing harnesses the power of genome editing tools to correct erroneous genes associated with disease pathology. To bring the CRISPR/Cas9 tool from the bench to the bedside, a critical hurdle is the safe and efficient delivery of this nucleic acid tool to the desired type of cells in patients. This review discusses the use of natural carriers, extracellular vesicles (EVs), in particular exosomes, to fill the gap. Exosomes are lipid-containing nanovesicle released by various types of cells to mediate cell-cell communications. Their inherent long-distance transportation capability, biocompatibility, and engineerability have made EVs potential vehicles for delivering therapeutic drugs. We summarize the recent progress of harnessing exosomes as delivery vehicles for the CRISPR/Cas system to achieve therapeutic gene editing for disease treatment, with a focus on various strategies to achieve selective delivery to a particular type of cell and efficient packaging of the genome editing tools in the vesicles. Critical issues and possible solutions in the design and engineering of the targeting vehicles are highlighted. Taken together, we demonstrate EV/exosome-mediated packaging of the nucleic acid/protein tools and the cell/tissue-targeted delivery to be a viable way towards the clinical translation of the CRISPR/Cas9 technology.     

Mitochondrial Genome Editing to Treat Human Osteoarthritis—A Narrative Review

Osteoarthritis (OA) is a severe, common chronic orthopaedic disorder characterised by a degradation of the articular cartilage with an incidence that increases over years. Despite the availability of various clinical options, none can stop the irreversible progression of the disease to definitely cure OA. Various mutations have been evidenced in the mitochondrial DNA (mtDNA) of cartilage cells (chondrocytes) in OA, leading to a dysfunction of the mitochondrial oxidative phosphorylation processes that significantly contributes to OA cartilage degeneration. The mitochondrial genome, therefore, represents a central, attractive target for therapy in OA, especially using genome editing procedures. In this narrative review article, we present and discuss the current advances and breakthroughs in mitochondrial genome editing as a potential, novel treatment to overcome mtDNA-related disorders such as OA. While still in its infancy and despite a number of challenges that need to be addressed (barriers to effective and site-specific mtDNA editing and repair), such a strategy has strong value to treat human OA in the future, especially using the groundbreaking clustered regularly interspaced short palindromic repeats (CRIPSR)/CRISPR-associated 9 (CRISPR/Cas9) technology and mitochondrial transplantation approaches.     

CRISPR/Cas-Mediated Resistance against Viruses in Plants

CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.     

Single-Strand Annealing in Cancer

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.