Programmable RNA targeting with clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas7-11 in zebrafish embryos and mammalian cells
Findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.
Abstract
The CRISPR/Cas7-11 system is a recently characterized RNA targeting tool that exhibits low toxicity, minimal off-target effects, and reduced collateral RNA cleavage. While Cas7-11 has been recently tested in zebrafish embryos as ribonucleoprotein (RNP) complexes, its use as mRNA–single guide RNA (sgRNA) formulations and its application in human embryonic stem cells (hESCs) have not been explored.
In this study, we evaluated the efficiency and specificity of
Cas7-11
in zebrafish embryos and hESCs. In zebrafish,injection ofCas7-11and sgRNA resulted in evident reductions of target transcripts, accompanied by clear phenotypic outcomes. Exogenous
EGFP
mRNA levels were reduced to 31.90%, while endogenous tbxta was reducedto 19.89%, and tyrosinase to 33.18%, respectively, resulting in the no-tail phenotype and reduced pigmentation. Theno-tail phenotype was partially rescued by mRNA overexpression. RNA sequencing confirmed minimal off-target effects. Moreover, Cas7-11 decreased exogenous
Gaussia luciferase
mRNA in human embryonic and HEK293T cells, to 23.52% and to 63.75%, respectively, and correspondingly reduced luciferase activity, without collateral cleavage activity. Targeting pluripotency factor genes, OCT4 and SOX2, in hESCs decreased their mRNA levels and induced differentiation.
Collectively, these findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.
Primordial germ cells (PGCs) are a unique platform for heritable gene editing in avian species, because they allow easy isolation, culture, and can then be reintroduced into the host. CRISPR/Cas9 technologies have advanced avian genome editing by enabling targeted editing of genes and traits for production, health, reproduction, and welfare. This review critically evaluates genome-editing tools in avian PGCs, including CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), base editing, and prime editing. Germline transmission efficiency, suitability, precision, and heritability are compared. Efficiency, cytotoxicity, and translational feasibility of delivery strategies, including viral vectors, electroporation and the use of ribonucleoproteins are assessed. Applications of these techniques in chickens are for muscle growth via myostatin (MSTN) gene disruption, viral resistance via editing of the sodium/hydrogen exchanger 1 (NHE1) gene, and control of male and female ratios through modification of sex determination genes. Additionally, applications in biopharmaceutical protein production and animal biodiversity conservation are also explored. Despite advances, some limitations remain, including low efficiency of homology-directed repair, off-target effects, mosaicism, and variability in transmission through the germ line. Current evidence demonstrates a significant lack of germline validation and scalability creating barriers to translate this potential into commercial avian production, especially, poultry breeding. Regulatory frameworks and their implications for food and commercialization are also discussed. Future research should prioritize precision editing, scalable delivery systems, and regulatory alignment to enable practical, ethical, and responsible implementation.
Mazhar Farooq, Dayong Han, Lei Zhang et al.· Poultry Science· 0 citations
Findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin/BMP network can generate twin-tail-like caudal morphology.
Huijuan Li, Xiaoying Zhang, Xiaowen Wang et al.· International Journal of Mol...· 0 citations
CRISPR-Cas9 ribonucleoprotein (RNP)-mediated genome editing has recently been established in the green seaweed Ulva. However, achieving precise and efficient targeted gene insertion remains challenging due to the low frequency of homology-directed repair (HDR) and suboptimal donor DNA design. In this study, we attempted to optimize a knock-in strategy by co-delivering Cas9 RNPs and donor DNA templates to target the highly expressed RbcS gene for EGFP insertion, while simultaneously disrupting the adenine phosphoribosyltransferase (APT) gene for robust selection. We compared the efficacy of single-stranded (ssDNA) versus double-stranded (dsDNA) donors with varying homology arm (HA) lengths. We found that ssDNA donors significantly outperformed dsDNA templates. Furthermore, 50-nt HAs were ineffective, while ssDNA donors with 300-nt HAs achieved the highest insertion efficiency. Sequence analysis revealed the loss of a donor-specific deletion, suggesting that Ulva utilizes synthesis-dependent strand annealing (SDSA) or mismatch repair pathways, rather than the microhomology-mediated mechanisms prevalent in Chlamydomonas. The APT-based co-targeting strategy effectively enriched the candidate population, enabling a discovery rate of approximately 3% for EGFP-positive strains among resistant individuals, achieving the first successful generation of a targeted double mutant in this species. Additionally, using tandem 2 A peptides (P2A-T2A) significantly improved ribosomal skipping efficiency compared to single 2 A systems, facilitating effective polycistronic expression. Collectively, this study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva, thereby expanding the genetic engineering toolkit for this macroalga.
K. Ichihara, Chikako Nagasato, T. Yamazaki et al.· Phycology Journal· 0 citations
Rationale Efficient gene activation or repression through programmable CRISPR-Cas9 has revolutionized molecular biology and drug development. Nonetheless, the currently available CRISPRa/i approaches are modestly potent and require multi-component delivery, which hampers the wide use of the technology in both research and therapy. Methods We developed a modular CRISPR-condensate platform by appending a multivalent RNA nanostar to the 3’ end of a single-guide RNA, producing a sgRNA-nanostar chimera that mediates phase separation at Cas9-bound genomic loci. The nanostar scaffold also contains MS2 stem-loops, which recruit MCP-tagged transcriptional effectors (VP64 for activation, KRAB for repression) to the condensate microenvironment at high local concentration. We examined condensate formation, genome targeting, and transcriptional output by using live-cell imaging, RT-qPCR, ChIP-seq, RNA-seq and CUT&Tag in HEK293T, HeLa, U-2 OS, MDA-MB-231, as well as human iPSC cell lines. Results The CRISPR-condensate design resulted in up to 50–100-fold target-gene activation, compared with 5–10-fold activation by direct VP64 fusion, and 20–30-fold transcriptional repression, compared with 3–5-fold repression by direct KRAB fusion, with high target specificity (12 versus 28 non-target differentially expressed genes assessed by RNA-seq). Orthogonal kissing-loop (KL) pairings enabled independent condensate systems for simultaneous activation and repression of multiplexed targets. Janus condensates containing both activating and repressive domains enabled bidirectional regulation at a single locus. The system requires delivery of only three independently expressible components—dCas9-NLS, an sgRNA-nanostar chimera bearing MS2 stem-loops (MS2SLs), and an MCP-fused effector (VP64-MCP for activation or KRAB-MCP for repression)—and showed minimal innate immune response and high cell viability. Conclusions The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation. This strategy makes biomolecular condensation a general principle for enhancing CRISPR gene regulation, opening up possibilities for functional genomics, cell engineering, and therapy development.
Aolin Li, Congcong Cao, Chunyan Yang et al.· Theranostics· 0 citations
Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA.
A. Udristioiu, Manole Cojocaru· Cancer Research· 0 citations
Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA.
A. Udristioiu, Manole Cojocaru· Clinical Cancer Research· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026
