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Clinical Milestone for CRISPR Medicines.
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Utilization of Miniature CRISPR-AsCas12f1 Nuclease for Efficient Genome Editing in Bacillus subtilis.
To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.
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An effective Caenorhabditis elegans CRISPR training module for high school and undergraduate summer research experiences in molecular biology.
Engaging in research experiences as a high school or undergraduate student interested in science, technology, engineering, and mathematics (STEM) is pivotal for their academic and professional development. A structured teaching framework can help cultivate a student's curiosity and passion for learning and research. In this study, an eight-week training program was created to encompass fundamental molecular biology principles and hands-on laboratory activities. This curriculum focuses on using clustered regularly interspaced short palindromic repeats (CRISPR) gene editing in the Caenorhabditis elegans model organism. Through pre- and post-program assessments, enhancements in students' molecular biology proficiency and enthusiasm for scientific exploration were observed. Overall, this training module demonstrated its accessibility and ability to engage inexperienced students in molecular biology and gene editing methodologies.
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Traditional Islamic approach can enrich CRISPR twins debate.
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Genome Editing and Pathological Cardiac Hypertrophy.
Three major genome editing tools, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat (CRISPR) systems, are increasingly important technologies used in the study and treatment of hereditary myocardial diseases. Germ cell genome editing and modification can permanently eliminate monogenic cardiovascular disease from the offspring of affected families and the next generation, although ethically controversial. Somatic genome editing may be a promising method for the treatment of hereditary cardiomyopathy various diseases for which gene knockout is favorable and can also treat people who are already ill, although there are currently some technical challenges. This chapter describes the application of genome editing in the experimental studies and treatment of hypertrophic cardiomyopathy as well as other cardiomyopathies.
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Tiny CRISPR molecular switch tool opens a new world.
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Improving CRISPR tools by elucidating DNA repair.
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Evaluation of CRISPR/Cas9 site-specific function and validation of sgRNA sequence by a Cas9/sgRNA-assisted reverse PCR technique.
The effective application of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system in biology, medicine and other fields is hindered by the off-target effects and loci-affinity of Cas9-sgRNA, especially at a genome-wide scale. In order to eliminate the occurrence of off-target effects and evaluate loci-affinity by CRISPR/Cas9 site-specific detection and screening of high-affinity sgRNA sequences, respectively, we develop a CRISPR/Cas9-assisted reverse PCR method for site-specific detection and sgRNA sequence validation. The detection method based on PCR can be used directly in the laboratory with PCR reaction conditions, without the need for an additional detection system, and the whole process of detection can be completed within 2 h. Therefore, it can be easily popularized with a PCR instrument. Finally, this method is fully verified by detecting multiple forms of site mutations and evaluating the affinity of a variety of sgRNA sequences for the CRISPR/Cas9 system. In sum, it provides an effective new analysis tool for CRISPR/Cas9 genome editing-related research. A CRISPR/Cas9-assisted reverse PCR method was developed for Cas9/sgRNA site-specific detection and sgRNA sequence validation. The technique detects target DNA in three steps: (1) target DNA is specifically cut by a pair of Cas9/sgRNA complexes; (2) the cleaved DNA is rapidly linked by T4 DNA ligase; (3) the ligated DNA is efficiently amplified by PCR (PCR or qPCR).
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Special Issue: Applications of CRISPR Technology in Virology 2018.
Precision genome engineering by CRISPR is a game-changing technology that originates from the study of virus-host interaction and promises to revolutionize virology and antiviral therapy [...].
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Now for the hard ones: is there a limit on CRISPR genome editing in crops?
This article comments on the following paper: Martín-Pizarro C, Triviño JC, Posé D. 2019. Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis. Journal of Experimental Botany 70, 885–895.
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