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Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos.
IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo.
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Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.
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Modified RNAs in CRISPR/Cas9: An Old Trick Works Again.
Old tricks, new dog: CRISPR/Cas9 is a powerful tool for gene editing that requires an endonuclease (Cas9) and RNA strands. It has been shown that chemical modification of the RNA structures, an approach that has been used to improve the efficiency of RNA interference, can also be applied to enhance the activity of CRISPR/Cas9 and reduce its off-target effects.
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mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology.
Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how "Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9" by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and "Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9" by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.
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Recent advances of Cas12a applications in bacteria.
Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. KEY POINTS: • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches.
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Genome editing: proceed with caution.
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Conditional and tissue-specific approaches to dissect essential mechanisms in plant development.
Reverse genetics approaches are routinely used to investigate gene function. However, mutations, especially in critical genes, can lead to pleiotropic effects as severe as lethality, thus limiting functional studies in specific contexts. Approaches that allow for modifications of genes or gene products in a specific spatial or temporal setting can overcome these limitations. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies has not only revolutionized targeted genome modification in plants but also enabled new possibilities for inducible and tissue-specific manipulation of gene functions at the DNA and RNA levels. In addition, novel approaches for the direct manipulation of target proteins have been introduced in plant systems. Here, we review the current development in tissue-specific and conditional manipulation approaches at the DNA, RNA, and protein levels.
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Repurposing CRISPR System for Transcriptional Activation.
In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most popular one for genome editing. When the nuclease domains of Cas9 protein are mutated into deactivated form (dCas9), CRISPR/dCas9 still retains the ability to bind the targeted DNA sequence, but loses the endonuclease cleavage activity. Taking advantage of the characteristics of this engineered nuclease inactive Cas9, the CRISPR/dCas system has been repurposed into versatile RNA-guided, DNA-targeting platforms, such as genome imaging, gene regulation, and epigenetic modification. Specifically, fusion of dCas9 with activation domains allows specific and efficient transcriptional activation on a genome-wide scale among diverse organisms. The purpose of this chapter is to review most important the recently published literature on CRISPR/dCas9-based transcriptional activation systems. Compared with the conventional approaches for enhancement of the expression of specific genes of interest, CRISPR/Cas9-based system has emerged as a promising technology for genome regulation, allowing specificity, convenience, robustness, and scalability for endogenous gene activation.
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Computation empowers CRISPR discovery and technology.
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CRISPR-Assisted Probiotic and In Situ Engineering of Gut Microbiota: A Prospect to Modification of Metabolic Disorders.
The gut microbiota, a substantial group of microorganisms residing in the human body, profoundly impacts various physiological and pathological mechanisms. Recent studies have elucidated the association between gut dysbiosis and multiple organ diseases. Gut microbiota plays a crucial role in maintaining gastrointestinal stability, regulating the immune system and metabolic processes not only within the gastrointestinal tract but also in other organs such as the brain, lungs, and skin. Dysbiosis of the gut microbiota can disrupt biological functioning and contribute to the development of metabolic disorders. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) modules are adaptive immune systems in numerous archaea and bacteria. CRISPR/Cas is a versatile gene-editing tool that enables modification of the genome in live cells, including those within the gut microbiota. This technique has revolutionized gene editing due to its simplicity and effectiveness. It finds extensive applications in diverse scientific arenas, facilitating the functional screening of genomes during various biological processes. Additionally, CRISPR has been instrumental in creating model organisms and cell lines for research purposes and holds great potential for developing personalized medical treatments through precise genetic alterations. This review aims to explore and discuss the possibilities of CRISPR/Cas and the current trends in using this technique for editing gut microbiota genes in various metabolic disorders. By uncovering the valuable potential of CRISPR/Cas in modifying metabolic disorders through the human gut microbiota, we shed light on its promising applications.
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