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High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase.
Base editors (BEs), covalent fusions of a cytosine or adenine deaminase with a nuclease-impaired CRISPR protein, mediate site-specific conversion of C:G to T:A (CBEs) or A:T to G:C (ABEs) in the genome. Existing BEs modify all cytosines or adenines within the editing window, which limits their precision. Here we engineer nucleotide and context specificity of the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to pinpoint cytosine editing. Strategically sampling multiple nucleic-acid-recognition hotspots through directed evolution, we develop 16 TadA-derived NCN-specific deaminases that cover every possible -1 and +1 context for a target cytosine, providing on-demand deaminase choices for editor customization. We apply these variants to (1) correct disease-associated T:A-to-C:G transitions documented by ClinVar, achieving greater accuracy than conventional CBEs in 81.5% of cases, and (2) model two cancer-driver mutations-KRASG12D (ACC) and TP53R248Q (CCG)-in vitro. Our approach offers a general strategy to access precise base editors for potential clinical applications.
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In vivo analysis of renal epithelial cells in zebrafish.
The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.
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Establishment of CRISPR/Cas9 mediated targeted mutagenesis in hop (Humulus lupulus).
The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.
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Computational Tools and Resources Supporting CRISPR-Cas Experiments.
The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.
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CRISPR/Cas9-Based Genome Editing of Transcription Factor Genes in Marchantia polymorpha.
Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA. Double-strand breaks sometimes induce insertions and deletions at the target site, leading to frameshift mutations of TF genes. In this chapter, we describe a detailed protocol for the targeted mutagenesis of TFs using the CRISPR/Cas9 system, specifically in the case of Marchantia polymorpha, an emerging model plant for functional genomics. The CRISPR/Cas9 system is highly versatile for targeting genomic sequences because only an alteration of the gRNA sequence is needed to change target sequences. The labor and cost required to establish genome-edited lines are low enough that multiple mutants of TF genes can be generated in one laboratory. The CRISPR/Cas9-based genome editing technique consists of four steps: (1) gRNA design; (2) vector construction; (3) transformation; and (4) isolation of genome-edited lines. This manuscript focuses mainly on the strategy of gRNA design, the workflow for off-target searches, and the selection and identification of genome-edited lines by genotyping. Although we describe a protocol for M. polymorpha, the basic strategy of generating genome-edited lines of TF genes should be applicable widely to other plants.
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Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells.
BACKGROUND: The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. RESULTS: The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). CONCLUSIONS: This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.
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Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.
In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
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Intellectual property education exemplified by the patents on the CRISPR/Cas9 system.
With the accelerated globalization of the world economies, the role of intellectual property in the the competition is increasingly important. The universities are important base to instill the intellectual property awareness to the young generation. However, current model of intellectual property education cannot meet the needs of undergraduates. In this paper, we take the first patent issued for CRISPR/Cas9 system as a teaching example, and together with personal teaching experience in biomedicine related intellectual property, we propose a new way for intellectual property education which consists of two stages: enlightenment stage and in-depth training stage. In the former stage, we integrate the intellectual property education with the basic major courses. In the latter stage, students are encouraged to devote into intellectual property related career. This model can somehow solve the the current shortage of qualified teachers for biotechnology related intellectual property education and will facilitate the popularization of intellectual property in college students. Since genetics plays a pivotal role in biomedicine, this effort is illustrated by the novel genome editing technology based on the CRISPR/Cas9 system, which is one hotspot of recent studies. The trajectory of CRISPR/Cas9 from basic microbial genetics discovery to major tools for genome editing exeplified the essence of biomedicine related intellectual property education.
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CRISPR-Cas9 genome editing: technological advances, delivery strategies and precision engineering of primary cells for therapeutic application in genetic disorders and diabetes mellitus.
The CRISPR-Cas9 system has revolutionized modern life sciences, driving a paradigm shift in biomedical research and becoming an indispensable tool in molecular biology due to its remarkable precision, efficiency, and simplicity. Originating from a bacterial adaptive immune mechanism, CRISPR-Cas9 has evolved rapidly, providing a versatile framework for manipulating genetic material and addressing a wide spectrum of human diseases. Recent progress includes the discovery of novel Cas orthologs and the rational engineering of Cas9 variants to enhance editing fidelity, broaden target range, and minimize off-target effects. Structural and functional optimization of single-guide RNAs (sgRNAs) has further improved target binding affinity, stability, and Cas9-sgRNA complex formation, thereby increasing overall editing performance. The development of high-fidelity Cas9 derivatives and next-generation platforms such as base editors and prime editors has enabled precise single-nucleotide substitutions and small insertions or deletions without generating double-stranded DNA breaks. Advanced delivery systems-including viral vectors, lipid nanoparticles, and ribonucleoprotein electroporation-have facilitated efficient CRISPR-mediated editing across in vitro, ex vivo, and in vivo models. Remarkable therapeutic milestones have been achieved in treating monogenic disorders such as sickle cell disease, β-thalassemia, and cystic fibrosis, where long-term clinical benefits have been documented. Furthermore, CRISPR-Cas9 technology is redefining diabetes research by enabling precise modeling of disease mechanisms, uncovering molecular pathways involved in glucose homeostasis, and opening new avenues for cellular and gene-based therapies. This review highlights recent advances in CRISPR-Cas9-mediated genome editing, emphasizing breakthroughs in primary cell editing and the development of translational models for genetic diseases and diabetes mellitus.
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Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.
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