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SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling.
We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of "blocking" silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard "correction-only" strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.
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An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy.
The CRISPR/Cas9 system is a powerful genome editing tool that has the potential to be applied to a variety of biomedical applications. Despite the considerable potential of this gene editing technology, there are numerous safety concerns including the possibility of unpredictable off-target effects. The splicing process, which involves the removal of introns from pre-mRNA and the alignment of exons to produce mature transcripts, is a critical step in gene expression in most eukaryotes. In this study, we present a spliceosome-responsive CRISPR/Cas9 (SpC) editor that utilizes the splicing inhibitor pladienolide B (PB) to regulate pre-mRNA splicing and control the expression of the anti-CRISPR protein AcrIIA4, thereby modulating the activity of the Cas9 nuclease. This approach allows for precise regulation of the gene editing process, thereby effectively mitigating off-target effects. The reliability and robustness of the SpC editor were demonstrated through in vitro and in vivo bioluminescence imaging. Furthermore, a dual-target sgRNA was designed to target the diphtheria toxin A gene, resulting in apoptosis induction and growth inhibition of tumor cells across various types of cancer cells. Our results indicate that this SpC editor has the capacity to precisely regulate tumor cell growth, thus providing new insights and significant implications for cancer gene therapy.
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Engineering a robust Cas12i3 variant-mediated wheat genome editing system.
Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and its less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3-5M-mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3-5M-mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3-5M increased the gene editing efficiencies by up to 1.34-fold and 3.87-fold, compared to Cas12i3-5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt-T5E-Cas12i3-5M could enhance the editing efficiency by 1.20- to 1.33-fold and 4.05- to 7.95-fold in wheat stable lines compared to Opt-Cas12i3-5M and Opt-Cas12i3, respectively, due to progressive 5'-end resection of the DNA strand at the cleavage site with increased deletion size. The Opt-T5E-Cas12i3-5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt-T5E-Cas12i3-5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.
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[Prediction of CRISPR/Cas9 off-target activity using multi-scale convolutional neural network].
Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) is a new generation of gene editing technology, which relies on single guide RNA to identify specific gene sites and guide Cas9 nuclease to edit specific location in the genome. However, the off-target effect of this technology hampers its development. In recent years, several deep learning models have been developed for predicting the CRISPR/Cas9 off-target activity, which contributes to more efficient and safe gene editing and gene therapy. However, the prediction accuracy remains to be improved. In this paper, we proposed a multi-scale convolutional neural network-based method, designated as CnnCRISPR, for CRISPR/Cas9 off-target prediction. First, we used one-hot encoding method to encode the sgRNA-DNA sequence pair, followed by a bitwise or operation on the two binary matrices. Second, the encoded sequence was fed into the Inception-based network for training and evaluating. Third, the well-trained model was applied to evaluate the off-target situation of the sgRNA-DNA sequence pair. Experiments on public datasets showed CnnCRISPR outperforms existing deep learning-based methods, which provides an effective and feasible method for addressing the off-target problems.
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Gene Editing of Primary Rhesus Macaque B Cells.
B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.
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A versatile tool for gene editing in the diatom Thalassiosira pseudonana.
Diatoms are major contributors to marine primary production, with the centric diatom Thalassiosira pseudonana a powerful model for understanding biogeochemical processes including carbon fixation and silicification. While there are molecular tools available for fluorescent protein (FP) tagging and CRISPR/Cas9 genome editing in T. pseudonana, these require the delivery of multiple vectors or have limited versatility. Additionally, scarless endogenous tagging, that results in a FP fusion expressed from its native genomic location, has yet to be developed. Here, we describe a versatile modular Golden Gate-based toolkit for T. pseudonana that through the delivery of a single-episome via bacterial conjugation enables: (i) FP tagging, (ii) dual FP tagging, (iii) CRISPR/Cas9 genome editing, (iv) simultaneous FP tagging with gene editing, and (v) scarless endogenous FP tagging. We further expand the available parts for T. pseudonana by validating 3 additional FPs and 2 untested promoter/terminator pairs. We demonstrate the versatility of our system by knocking out diatom pyrenoid component 1 (DPC1) while simultaneously GFP tagging the rbcS (Rubisco small subunit), and by endogenously GFP tagging the bestrophin-like protein BST2. While DPC1 knockout does not result in a major pyrenoid structural defect due to unperturbed rbcS-GFP localization to the pyrenoid, we confirm that BST2 localizes to the pyrenoid and exhibits increased fluorescence under low CO2-supporting a role in diatom carbon fixation. Our developed genetic tools provide a robust framework for exploring cellular processes in diatoms, accelerating routine studies and enabling systematic, quantitative and large-scale studies.
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Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct.
The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species' triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.
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Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing.
Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.
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CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis.
Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+ signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+ release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+ signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.
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Keep off-target effects in focus.
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