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Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277.
BACKGROUND: CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi. RESULTS: In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci. CONCLUSIONS: The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.
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Therapeutic genome editing in cardiovascular diseases.
During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.
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Application of CRISPR/Cas9 to Autophagy Research.
The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.
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CRISPR-based genetic tools for the study of host-microbe interactions.
CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.
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Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells.
Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.
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Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq.
Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.
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Seed sequences mediate off-target activity in the CRISPR-interference system.
The CRISPR interference (CRISPRi) system is a powerful tool for selectively and efficiently silencing genes in functional genomics research applications. However, its off-target activity has not been systematically investigated. Here, we utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library to investigate the presence of off-target activity and its effects on gene expression. Our findings suggest that off-target effects in CRISPRi are quite pervasive and have direct and indirect impacts on gene expression. Most of the identified off-targets can be accounted for by complementarity of the protospacer adjacent motif (PAM)-proximal genomic sequence with the 3' half of the sgRNA spacer sequence, the seed sequence. We also report that while the stability of off-target binding is primarily driven by the PAM-proximal seed sequences, variations in the length of these seed sequences and the degree of mismatch tolerance at various positions can differ across different sgRNAs.
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Construction of the PG-deficient mutant of Fusarium equiseti by CRISPR/Cas9 and its pathogenicity of pitaya.
Fusarium is an important plant pathogen and many cell wall-degrading enzymes (CWDEs) are produced in Fusarium-infected plant tissues. To investigate the role of CWDEs in the pathogenicity of pitaya pathogen, we isolated a Fusarium equiseti strain from the diseased pitaya fruit and the activities of CWDEs were determined. The higher polygalacturonase (PG) activity was confirmed both in vitro and vivo. Aiming at the PG gene, the CRISPR/Cas9 system of F. equiseti was constructed and optimized for the first time. Through the process of microhomology-mediated end joining, the flanking region containing 30 bp was used to mediate the homologous recombination of Cas9 double-strand breaks, and the PG gene knockout mutants were obtained by protoplast transformation. Through the phenotypic and pathogenicity experiments of the wild-type strain and mutant strain, the results showed that the colony growth rate and spore production of the strain without the PG gene decreased to some extent, and the lesion diameter and the degree of pericarp cell damage decreased, which showed that the CRISPR/Cas9 system could be used in F. equiseti and PG enzyme and can play a significant role in the interaction between F. equiseti and pitaya fruit.
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U.S. consumers' processing of information about CRISPR-edited pork products.
The commercialization of CRISPR gene-edited pork is advancing rapidly, following the U.S. Food and Drug Administration's approval of gene-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS). As these products move closer to market entry, understanding how consumers seek, process, and avoid information about them is critical for developing effective communication strategies. Guided by the Risk Information Seeking and Processing (RISP) model, this study examined factors influencing information seeking, information avoidance, and information processing related to CRISPR-edited pork products among U.S. consumers (n = 2,006). Results show higher information sufficiency thresholds were associated with greater information seeking and lower information avoidance. Information seeking was strongly and positively correlated with systematic processing. Relevant channel beliefs and perceived information gathering capacities were positively associated across communication channels, suggesting the need for integrated communication approaches. Relevant channel beliefs for news media and social media were positively associated with information seeking, while stronger relevant channel beliefs for Extension were associated with lower information seeking. Respondents with some college education reported higher information seeking than those with only a high school diploma or GED, while older adults and individuals with higher education levels reported lower information avoidance. Results also showed that respondents exhibited high intentions to seek information and low tendencies to avoid information, suggesting openness to learning about CRISPR-edited pork. Participants also reported engaging more in systematic processing than heuristic processing, indicating a preference for careful and analytical evaluation of information. Findings highlight the importance of audience segmentation, multi-channel communication strategies, and evidence-based messaging to support informed public engagement with CRISPR-edited food technologies.
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Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9.
Natural killer cells are potent cytotoxic lymphocytes specialized in recognizing and eliminating transformed cells, and in orchestrating adaptive anti-tumour immunity. However, NK cells are usually functionally exhausted in the tumour microenvironment. Strategies such as checkpoint blockades are under investigation to overcome NK cell exhaustion in order to boost anti-tumour immunity. The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene-editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene-editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells. The current review discusses feasible approaches to deliver the CRISPR/Cas9 systems, as well as potential strategies in gene-editing for NK cell immunotherapy for cancers.
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