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Microhomology-assisted scarless genome editing in human iPSCs.
Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
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Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.
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Genetic and Molecular Quality Control of Genetically Engineered Mice.
Genetically engineered mice are used as avatars to understand mammalian gene function and develop therapies for human disease. During genetic modification, unintended changes can occur, and these changes may result in misassigned gene-phenotype relationships leading to incorrect or incomplete experimental interpretations. The types of unintended changes that may occur depend on the allele type being made and the genetic engineering approach used. Here we broadly categorize allele types as deletions, insertions, base changes, and transgenes derived from engineered embryonic stem (ES) cells or edited mouse embryos. However, the methods we describe can be adapted to other allele types and engineering strategies. We describe the sources and consequ ences of common unintended changes and best practices for detecting both intended and unintended changes by screening and genetic and molecular quality control (QC) of chimeras, founders, and their progeny. Employing these practices, along with careful allele design and good colony management, will increase the chance that investigations using genetically engineered mice will produce high-quality reproducible results, to enable a robust understanding of gene function, human disease etiology, and therapeutic development.
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CTCF driven TERRA transcription facilitates completion of telomere DNA replication.
Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.
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abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas9 genome editing.
SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58 875 004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing. AVAILABILITY AND IMPLEMENTATION: The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.
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A Practical Guide to Genome Editing Using Targeted Nuclease Technologies.
Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.
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Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation.
Mycoplasmas are successful pathogens that cause debilitating diseases in humans and various animal hosts. Despite the exceptionally streamlined genomes, mycoplasmas have evolved specific mechanisms to access essential nutrients from host cells. The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of the virulence factors of pathogenic species and mechanisms to access nutrients. This review summarizes several strategies for editing of mycoplasma genomes, including homologous recombination, transposons, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and synthetic biology. In addition, the mechanisms and features of different tools are discussed to provide references and future directions for efficient manipulation of mycoplasma genomes.
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CRISPR-driven innovations in rice (Oryza sativa L.) breeding: precision development of male sterile lines.
Male sterility is a vital trait exploited in hybrid seed production to boost crop yield and improve quality. In rice, different male sterility systems have been developed, significantly advancing the production of high-yielding hybrids. The three main types of male sterility in rice are cytoplasmic male sterility (CMS), photoperiod-sensitive genic male sterility (PGMS), and genic male sterility (GMS). Among these, CMS is the most widely used, arising from interactions between mitochondrial (cytoplasmic) and nuclear genes, resulting in pollen dysfunction. PGMS, on the other hand, is influenced by environmental cues such as day length and temperature, while GMS is attributed to mutations in specific nuclear genes affecting anther or pollen development. A thorough understanding of the genetic and molecular mechanisms underlying these systems is essential for efficient hybrid rice breeding. CMS lines are typically crossed with maintainer and restorer lines carrying fertility-restoring genes to produce fertile F₁ hybrids. Recent advancements in molecular biology, genomics, and genome editing technologies have accelerated the development of novel male-sterile and fertility-restoring lines, thereby enhancing the precision and scalability of hybrid breeding programs. These innovations are not only expanding the genetic base of hybrid rice but also making the production process more sustainable. As global food demand rises alongside climate uncertainties, the strategic use of male sterility in rice breeding holds immense potential for improving agricultural productivity.
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Circular Guide RNA for Improved Stability and CRISPR-Cas9 Editing Efficiency in Vitro and in Bacteria.
Due to its intrinsic RNA properties, guide RNA (gRNA) is the least stable component of the CRISPR-Cas9 complex and is a major target for modification and engineering to increase the stability of the system. While most strategies involve chemical modification and special processes, we created a more stable gRNA with an easy-to-use biological technique. Since circular RNAs are theoretically immune to all RNA exonucleases, we attempted to construct a circular gRNA (cgRNA) employing the autocatalytic splicing mechanism of the RNA cyclase ribozyme. First, the formation of the cgRNA, which has a length requirement, was optimized in vivo in E. coli cells. It was found that a cgRNA with an insert length of 251 bp, designated 251cgRNA, was functional. More importantly, cgRNA increased the editing efficiency of the tested base editors relative to normal linear gRNA. The cgRNAs were more stable in vitro under all tested temperature conditions and maintained their function for 24 h at 37 °C, while linear gRNAs completely lost their activity within 8 h. Enzymatically purified 251cgRNA demonstrated even higher stability, which was obviously presented on gels after 48 h at 37 °C, and maintained partial function. By inserting a homologous arm into the 251cgRNA to 251HAcgRNA cassette, the circularization efficiency reached 88.2%, and the half-life of 251HAcgRNA was 30 h, very similar to that of purified 251cgRNA. This work provides a simple innovative strategy to greatly increase the stability of gRNA both in vivo in E. coli and in vitro, with no additional cost or labor. We think this work is very interesting and might revolutionize the form of gRNAs people are using in research and therapeutic applications.
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Generation of human induced pluripotent stem cell line carrying SCN5AC2204>T Brugada mutation (MUSli009-A-1) introduced by CRISPR/Cas9-mediated genome editing.
Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.
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