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Harnessing bacterial immunity: CRISPR-Cas system as a versatile tool in combating pathogens and revolutionizing medicine.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has emerged as an adaptable instrument for several uses. The CRISPR-Cas system employs Cas proteins and programmable RNA molecules to guide the recognition and cleavage of specific DNA regions, permitting accurate genome editing. It is derived from the bacterial immune system and allows for accurate and efficient modification of DNA sequences. This technique provides unparalleled gene editing, control, and precise alteration opportunities. This review aims to offer a comprehensive update of the core concepts of the CRISPR-Cas system and recent progress, while also providing an overview of the significant applications in diverse fields such as microbiology and medicine. The CRISPR-Cas9 gene editing technique has facilitated substantial advancements in comprehending gene function, simulating diseases, and creating innovative therapeutics. CRISPR-based therapeutics present a hopeful prospect for addressing intricate ailments, including genetic disorders, malignancies, and infectious diseases, as they serve as viable substitutes for conventional pharmaceuticals. In microbiology, this method serves as a diagnostic and therapeutic tool that proves highly efficient in eliminating bacteria that have developed resistance to various antibiotics. Despite its significant potential, CRISPR encounters ethical, safety, and regulatory obstacles that necessitate meticulous deliberation. Concerns regarding off-target effects, poor delivery to target tissues, and unwanted side effects emphasize the necessity to thoroughly examine the technology. It is necessary to balance the advantages and difficulties CRISPR presents. Consequently, more rigorous preclinical and clinical experiments are essential before using it in humans.
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Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems.
We developed simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are co-expressed from a single Pol II promoter, without any additional processing machinery. Our SSTU systems are easy to construct and effective in mediating multiplex genome editing.
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CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.
The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.
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Special Issue on the Chemical Biology of CRISPR.
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CRISPR Empowers Tree Bioengineering for a Sustainable Future.
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CRISPR applications in microbial World: Assessing the opportunities and challenges.
Genome editing has emerged during the past few decades in the scientific research area to manipulate genetic composition, obtain desired traits, and deal with biological challenges by exploring genetic traits and their sequences at a level of precision. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing tool has offered a much better understanding of cellular and molecular mechanisms. This technology emerges as one of the most promising candidates for genome editing, offering several advantages over other techniques such as high accuracy and specificity. In the microbial world, CRISPR/Cas technology enables researchers to manipulate the genetic makeup of micro-organisms, allowing them to achieve almost impossible tasks. This technology initially discovered as a bacterial defense mechanism, is now being used for gene cutting and editing to explore more of its dimensions. CRISPR/Cas 9 systems are highly efficient and flexible, leading to its widespread uses in microbial research areas. Although this technology is widely used in the scientific community, many challenges, including off-target activity, low efficiency of Homology Directed Repair (HDR), and ethical considerations, still need to be overcome before it can be widely used. As CRISPR/Cas technology has revolutionized the field of microbiology, this review article aimed to present a comprehensive overview highlighting a brief history, basic mechanisms, and its application in the microbial world along with accessing the opportunities and challenges.
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High-Throughput Approaches to Pinpoint Function within the Noncoding Genome.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.
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CRISPR-based diagnostics for infectious diseases: mechanisms, advancements and clinical transformation prospects.
Infectious diseases continue to pose significant global public health challenges, necessitating the development of rapid, sensitive, specific, and field-deployable diagnostic platforms. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) has revolutionized genome editing and concurrently enabled a new generation of molecular diagnostic tools. Leveraging the inherent trans-cleavage activities of Cas enzymes, platforms such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) have emerged, combining target recognition precision with reporter systems to achieve ultra-sensitive detection of pathogen-specific nucleic acids. This review systematically examines the mechanistic foundations of CRISPR diagnostics, synthesizes recent advancements in infectious disease applications, evaluates their advantages in sensitivity, specificity, operational simplicity, and multiplexing capacity, and critically analyzes current implementation barriers and future translational pathways.
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Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.
CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.
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Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9.
Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.IMPORTANCE In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.
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