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Comprehensive assessment of activity, specificity, and safety of hypercompact TnpB systems for gene editing.
BACKGROUND: As the ancestor of CRISPR-Cas12 nucleases, TnpB represents the most compact gene editing tool currently available. Recent studies have identified multiple TnpB systems with gene editing activity in mammalian cells, and the potential of TnpB in treating diseases has been demonstrated in animal models. However, the editing characteristics of various TnpB systems, comparable to CRISPR tools, require more extensive investigation. RESULTS: Using a standardized evaluation framework, we conduct a thorough analysis of the editing properties of four TnpB variants alongside representative Cas12 and Cas9 tools applications. Overall, TnpBs exhibit intermediate editing activity and safety profiles among all tested systems, with ISYmu1 TnpB demonstrating a good performance in both editing activity and specificity. Considering its compact size, potent editing efficiency and high specificity, ISYmu1 TnpB represents a promising candidate for gene therapy. CONCLUSIONS: By comprehensively analyzing genome editing outcomes, we characterize TnpB systems for genome editing and identify ISYmu1 TnpB as an optimal miniature RNA-guided genome editors with balanced performance, highlighting its potential for therapeutic applications.
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Emerging applications of CRISPR/Cas9 gene editing technology in reversing drug resistance in oral squamous cell carcinoma.
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Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting.
Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knock-in/knock-out experiments, demonstrating broad utility and reproducibility. For additional details on the use and execution of this protocol, please refer to Patel et al.1.
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Gene Editing, Enhancing and Women's Role.
A recent article on the front page of The Independent (September 18, 2015) reported that the genetic 'manipulation' of IVF embryos is to start in Britain, using a new revolutionary gene-editing technique, called Crispr/Cas9. About three weeks later (Saturday 10, October 2015), on the front page of the same newspaper, it was reported that the National Health Service (NHS) faces a one billion pound deficit only 3 months into the new year. The hidden connection between these reports is that gene editing could be used to solve issues related to health care allocation. Improving the health of future generations might coincide with public health goals; it might improve the health of individuals and communities, and, if successful, might be seen as a public good. However, enhancing future generations will require In Vitro Fertilisation and Pre-implantation Genetic Diagnosis. Remarkably, the necessary involvement of women in an enhancing scenario has not been discussed by its proponents. The present discourse on moral obligations of future generations, although not referring to women, seems to imply that women might be required, morally, if not legally, to reproduce with IVF. Enhancing future generations will be gendered, unless the artificial womb is developed. These are challenging issues that require a wider perspective, of both women and men. Despite the lack of a unified feminist conclusion in the discussions about the merits and risks of human genome modification, there is an urgent need to clarify the role of women in this scenario.
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Heritable Virus-Induced Genome Editing (VIGE) in Nicotiana attenuata.
The CRISPR/Cas9 system is an extremely powerful tool for targeted mutagenesis in plants. However, plant genome editing relies on the labor-intensive plant regeneration method for generating gene-edited plants. To overcome this bottleneck, several virus-induced genome editing (VIGE) techniques have been developed. The VIGE system aims to induce targeted mutations in germ cells without plant regeneration. However, due to the delivery issues of a large Cas9 protein, scientists focus on developing a virus-mediated delivery system for guide RNA into Cas9-overproducing plants. Here, we describe how to induce heritable targeted mutations in a non-model plant, Nicotiana attenuata, using VIGE system. This method will be applied for manipulating the target genes in any plants that scientists are interested in.
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Disruption of mstn Gene by CRISPR/Cas9 in Large Yellow Croaker (Larimichthys crocea).
The large yellow croaker (Larimichthys crocea) plays an economically vital role in the marine aquaculture in China. Suffering from infection of bacteria and protozoon, effect of extreme weather and stress from high-density farming, genome editing is thought to be an important tool applied to L. croea for enhancing commercial traits such as growth rate, disease resistance, and nutrition component. In this study, we identified two mstn genes in L. croea and investigated the different phylogenetic clades, gene structures, and conserved syntenic relationships. To obtain fast-growing large yellow croaker, we specially selected two validated targets for mstnb knockout, which was homologous to mammalian myostatin gene (MSTN) and downregulated skeletal muscle growth and development. Five significant mutation types were generated in two mosaic mutants by transferring specific CRISPR/Cas9 RNPs (ribonucleoprotein) into the one-cell fertilized embryos based on CRISPR/Cas9 technology. Subsequently, we also elucidated the obstacles and possible measures to improve the success rate of inducing modified large yellow croaker. Our results would provide valuable method and reference for facilitating genome editing programs of the large yellow croaker in the future.
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Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.
Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.
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[Application and optimization of CRISPR/Cas system in bacteria].
Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.
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Genomic and Transcriptomic Analyses of Prime Editing Guide RNA-Independent Off-Target Effects by Prime Editors.
Prime editors (PEs) were developed to induce versatile edits at a guide-specified genomic locus. With all RNA-guided genome editors, guide-dependent off-target (OT) mutations can occur at other sites bearing similarity to the intended target. However, whether PEs carry the additional risk of guide-independent mutations elicited by their unique enzymatic moiety (i.e., reverse transcriptase) has not been examined systematically in mammalian cells. Here, we developed a cost-effective sensitive platform to profile guide-independent OT effects in human cells. We did not observe guide-independent OT mutations in the DNA or RNA of prime editor 3 (PE3)-edited cells, or alterations to their telomeres, endogenous retroelements, alternative splicing events, or gene expression. Together, our results showed undetectable prime editing guide RNA-independent OT effects of PE3 in human cells, suggesting the high editing specificity of its reverse-transcriptase moiety.
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CRISPR/Cas9-mediated editing of carotenoid biosynthesis genes alters carotenoid concentrations in kiwifruit.
BACKGROUND: CRISPR/Cas9 technology has garnered increasing attention for its simplicity and precision in genome editing, making it an indispensable tool for gene function research and crop genetic improvement. However, the inefficiency and time-consuming nature of genetic transformation continue to pose substantial challenges to its widespread application in woody plants. RESULTS: In this study, we developed a rapid and efficient Agrobacterium-mediated transformation system using petioles as explants for kiwifruit. Positive resistant seedlings were obtained within three months by inoculating on MS medium supplemented with 2.0 mg·L-1 6-benzylaminopurine (6-BA), 0.2 mg·L-1 naphthaleneacetic acid (NAA), and 10 mg·L-1 hygromycin, which was faster than using leaves as explants. Using this system, CRISPR/Cas9-mediated editing of phytoene desaturase (AcPDS) and ζ-carotene desaturase (AcZDS) achieved an editing efficiency of 20%. Transgenic kiwifruit lines with edited AcZDS exhibited a significant reduction in carotenoid content. CONCLUSIONS: Overall, we established an efficient Agrobacterium-mediated transformation system using petioles as explants, which is applicable for CRISPR/Cas9-mediated gene editing in kiwifruit, thereby facilitating functional gene studies and genetic improvement.
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