Programmable large-fragment genome integration using prime-editing-coupled serine integrases, such as PASTE and PASSIGE, remains constrained by the limited activity of wild-type Bxb1 (WT Bxb1) in mammalian cells. Recently, the AI-guided protein engineering framework EVOLVEpro enabled efficient identification of functional protein variants from limited experimental sampling and nominated epBxb1(T166R) as a highly active Bxb1 variant in episomal plasmid-to-plasmid recombination screens. Here, we systematically benchmarked epBxb1 (T166R) against WT Bxb1 and the previously validated high-activity eeBxb1 (V74A/E229K/V375I) variant in genome-integrated reporter and endogenous-locus PASSIGE assays at three well-characterized benchmarking loci (AAVS1, CCR5, and ACTB). epBxb1 showed reduced and variable transferability, with no detectable advantage over WT Bxb1 in the genome-integrated reporter system or at the AAVS1 safe-harbor locus, but produced modest, locus-dependent improvements at CCR5 and ACTB, with 1.89-fold and 1.58-fold increases, respectively. By contrast, eeBxb1 consistently showed superior activity, achieving up to ∼12-fold improvement over WT Bxb1. Four additional rounds of EVOLVEpro-guided optimization on the eeBxb1-BPNLS scaffold identified no reproducibly improved variant among 40 tested single-amino-acid substitutions. These results indicate reduced and genomic context-dependent transferability of EVOLVEpro-nominated Bxb1 variants and highlight the importance of application-matched genomic benchmarking when implementing AI-guided protein optimization for therapeutic genome writing.
Dog allergy is a prevalent IgE-mediated condition, with the salivary lipocalin Can f 1 accounting for the majority of dog-specific IgE reactivity in sensitized individuals. Existing strategies for managing dog allergy primarily rely on modulating host immune responses and do not address allergen production at its source. Here, we report the generation of genetically engineered dogs lacking Can f 1. Using CRISPR-Cas9 editing and somatic cell nuclear transfer, we produced two healthy beagle puppies carrying a frameshift mutation in exon 1 of the Can f 1 gene. Salivary and hair/dander analysis demonstrated the absence of Can f 1 protein in the edited dogs. Skin prick testing in a sensitized individual demonstrated robust IgE-mediated reactivity to control dog extracts but no detectable response to extracts from the edited dogs. Together, these findings demonstrate that targeted genetic knockout of the major dog allergen is compatible with canine development and can abolish the IgE-mediated allergic response, supporting the feasibility of a gene-based approach to reducing canine allergenicity.
CRISPR interference (CRISPRi) often uses single guide RNAs (sgRNAs) generated by in vitro transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.
Since the advent of Cas9-based CRISPR technologies in 2012, there has been a remarkable growth in genome editing research, literature, applications, and translational impact. Much of this research has been fueled by the global dissemination of CRISPR plasmids through nonprofit distribution by Addgene, as both a repository and distributor of enabling biological material. Recently, key milestones have been reached, with over 20,000 plasmids deposited by over 1,000 labs, being distributed over 300,000 times globally. The driving trends reflect multidimensional diversification in terms of effectors (Cas9, Cas12 and beyond), editing modalities (base editing, prime editing, epigenetic modification, CRISPRi/a), and deployment across phylogenetic groups (mammalian, bacterial, plant, yeast, insects, and more). Noteworthy, guide RNA and HDR templates account for the bulk of deposits, while cloning backbones are the most requested, and lentiviral plasmids comprise the majority of expression material. The data reflect a continued diversification of the CRISPR-based toolbox, robust interest in genome editing applications across the tree of life, maturation in terms of adoption, and rising relative distribution beyond the USA and China, with Addgene continuing to play a critical role in access to equitable and disruptive technologies.
Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors.
Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.
A major goal of clinically oriented CRISPR-Cas9-based applications is safe and effective in vivo gene editing (knockout or correction) with precise targeting. Substantial efforts have been devoted to the preclinical development of novel drug delivery platforms that enable efficient, targeted delivery. However, the immune responses induced by CRISPR-Cas9 treatment are often overlooked. Preexisting immunity to clinically relevant Cas9 proteins has already been established as a consequence of natural exposure to Cas9-bearing bacteria, which may implicate the safety and efficacy of CRISPR-Cas9-based therapies. Naturally, CRISPR-Cas9 therapies should be nonimmunogenic to avoid amplifying existing Cas9-specific immunity, especially cytotoxic T cell responses. Nonviral delivery systems, such as lipid nanoparticles (LNPs), are widely regarded as less immunogenic than more traditionally used viral vectors, even though LNPs are suitable as a vaccination platforms. In this study, we investigate the induction of SpCas9-directed immunity in C57BL/6 mice upon repeated dosing of LNPs encapsulating Cas9-coding mRNA in two different settings: (1) a vaccination-resembling setting using intramuscularly administered adjuvanted LNPs, and (2) a therapy-resembling setting using intravenously injected, liver-targeting LNPs. In both settings, Cas9-specific T cell responses were detected by evaluating increased total IFN-γ levels upon ex vivo restimulation of isolated splenocytes. However, undetectable Cas9-reactive antibodies induced in the therapeutic setting emphasize the discrepancy between humoral and cellular responses. To improve future monitoring of Cas9-specific T cell responses, we report six Cas9-derived epitopes recognized by CD8+ T cells, as well as a CD4+ T cell polypeptide carrying one of the CD8+ T cell epitopes that induced strong IFN-γ production ex vivo. This work is intended to facilitate the preclinical monitoring of Cas9-specific T cell responses in C57BL/6 mice and support the development of safe CRISPR-Cas9-based therapies.
Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, Armatimonadota, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.
Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to in vivo applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.
Hepatocyte transplantation (HTx) offers a safer, less invasive alternative to orthotopic liver transplantation for inherited metabolic liver diseases, especially in high-risk pediatric patients. Combining HTx with ex vivo gene editing is a promising autologous therapeutic strategy using the patient's cells. We investigated the feasibility of this approach by applying CRISPR-Cas9 gene knock-out to neonatal mouse hepatocytes and comparing their engraftment potential with that of mature adult cells in the Fah-/- mouse model of hereditary tyrosinemia type I (HT1). Electroporation-mediated gene editing did not significantly impair the ability of neonatal hepatocytes to engraft in vivo. Quantitative histological analysis revealed comparable liver repopulation levels between recipients of gene-edited neonatal cells and adult cells after hepatoxicity-mediated selection, providing a benchmark for electroporation-mediated gene editing in neonatal hepatocytes, and supporting the development of genetically corrected neonatal hepatocyte products as a crucial long-term or bridge-to-transplant therapeutic strategy for pediatric liver disease.
The correlation between CRISPR-Cas systems and plasmid-mediated bacterial antibiotic resistance is increasingly growing attention. However, currently no reports exist on the relationship between the CRISPR-Cas systems and the carriage of blaNDM or plasmids in E. coli. Here, molecular characterization and phylogenetic analysis of 639 E. coli isolated from humans in China were carried out. Depending on similarity in sequence, the type I-E CRISPR-Cas systems in E. coli can be grouped into two distinct clades, which we refer to for descriptive purposes within this study as the type I-E-S1 and I-E-S2, whereas the type I-E-S2 CRISPR-Cas system is further divided into I-E-S2a and I-E-S2b systems based on the presence of cas8e and cas11. ST167 (phylogroup A) and ST410 (phylogroup C) E. coli were observed bearing the type I-E-S1 and I-E-S2b systems, respectively. Compared with strains carrying the I-E-S1 type CRISPR-Cas system, the blaNDM carrying rate, the positive rate of IncX3 plasmid, and the positive rate of IncF plasmid of strains with the I-E-S2a type CRISPR-Cas system were evidently lower (p < 0.05); the blaNDM carrying rate and the positive rate of IncF plasmid of strains with the I-E-S2b type CRISPR-Cas system were evidently higher (p < 0.05). The blaNDM positive rate and IncF plasmid positive rate of strains carrying the I-E-S2a type CRISPR-Cas system were significantly lower than those of strains carrying the I-E-S2b type CRISPR-Cas system (p < 0.001). It proves that the I-E-S1, I-E-S2a, and I-E-S2b type CRISPR-Cas systems are beneficial for spreading blaNDM and IncX3 plasmids. We found significant differences in the cas gene sequences of the I-E-S1 and I-E-S2 type CRISPR loci. The type I-E CRISPR-Cas systems in E. coli isolated from Chinese sources are classified further for the first time, revealing their high correlation with blaNDM, phylogenetic groups, and multilocus sequence typing. This work paves the way for a deeper understanding of the role that CRISPR-Cas systems play in the rise of resistant E. coli ST167 and ST410.
CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.
