The CRISPR Journal

The development of the central nervous system (CNS) depends on tightly regulated gene expression programs that guide neural progenitor differentiation and neuronal subtype specification. The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of nervous system development, with a larval CNS composed of just over 200 neurons and sensory cells. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona, validated single-guide RNAs (sgRNAs) have yet to be validated for key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected using CRISPOR and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates with predicted Doench 16 and Doench Ruleset 3 (RS3) scores, observing a modest but improved correlation with RS3 predictions. Based on these results, we recommend considering both scoring algorithms, with RS3 potentially offering improved predictive value for Ciona.

Efficient genome writing in mammalian cells requires robust methods for integrating large DNA payloads. The previously described method mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) enables iterative, biallelic genome rewriting in mammalian stem cells with DNA payloads exceeding 100 kb. However, the lack of standardized vectors and certain technical constraints have limited its broader adoption. Here we present an improved plasmid toolkit designed to streamline the implementation of mSwAP-In. The toolkit includes two core vectors. pLP-TK (pCTC174) is a landing-pad plasmid compatible with Golden Gate assembly of genomic homology arms and supports both mSwAP-In and the recombinase-mediated cassette exchange method Big-IN. mSwAP-In MC2v2 (pKBA135) is a versatile Big DNA assembly and delivery vector that supports Gibson-based assembly and incorporates positive, negative, and fluorescent selection markers, as well as a backbone counterselection cassette to minimize unwanted plasmid integration. The vector architecture also enables propagation in yeast and bacterial hosts, inducible plasmid copy-number amplification in standard E. coli strains, and CRISPR/Cas9-mediated payload release through preinstalled guide RNA target sites. We further characterize the FCU1/5-FC counterselection system in mouse embryonic stem cells and define conditions that minimize its bystander toxicity. Finally, we provide a set of Cas9-gRNA expression plasmids optimized for common mSwAP-In applications. Together, these reagents constitute a standardized and experimentally validated toolkit that simplifies large-scale genome writing using mSwAP-In.

Targeted protein degradation using conditional degron tag (CDT) technology is a powerful method for rapidly degrading a protein of interest (POI) upon the addition of a degrader drug. A prerequisite for the temporally controlled degradation of an endogenous POI is the generation of homozygous knock-in cells with the degron tag integrated at either the N- or C-terminus of their gene loci. However, obtaining those homozygous knock-in cells often requires selecting many single-cell clones, as human cells typically exhibit low homology-directed repair (HDR) activities. Additionally, tagging a degron to an endogenous protein may inadvertently reduce protein expression, potentially affecting protein function even before the drug is administered. Here, we develop a method for generating degron-tagged knock-in cells that allows us to skip the laborious single-cell cloning. This method arose from our observation that most knock-in cells carry the degron tag only in one allele (heterozygous), while the other allele typically harbors a frameshift insertion/deletion. This observation allowed us to bypass the need for single-cell cloning. We validated our method by knocking in degron tags at the N-terminus of cytoplasmic dynein1 subunits or Adaptor Protein 2 (AP2) subunit. Our experiments confirmed the rapid degradation of these proteins and their functional inhibition in bulk cell populations. Additionally, to mitigate the reduced expression often associated with the degron tagging, we established a method to control expression levels by inserting a mini-promoter immediately upstream of the knock-in cassette. Our method simplifies the workflow for degron tag knock-ins and enhances the versatility of these valuable technologies.

Protospacer adjacent motifs (PAMs) and target-adjacent motifs (TAMs) are essential for target recognition by CRISPR-Cas and TnpB nucleases. Here we present TAMIPAMI, an efficient experimental and computational framework for rapid PAM/TAM identification. TAMIPAMI requires only a single control library and Cas or TnpB-treated library, simplifying experimental design, reducing cost, and providing greater accessibility for users. The platform interprets sequencing data with interactive visualizations and introduces a novel algorithm that determines the minimal exact set of degenerate IUPAC sequences describing the observed PAM/TAM patterns. Using this approach, we accurately recovered canonical motifs for several nucleases, including SpCas9, LbCas12a, AsCas12a, BrCas12b, Cas12i1, and AmaTnpB. TAMIPAMI is available as both a web application and command-line tool, ultimately providing an accessible and efficient platform for PAM/TAM discovery and characterization across CRISPR and OMEGA systems.

While CRISPR/Cas-based gene editing technologies have the potential to greatly advance crop breeding endeavours, intellectual property-related challenges can hinder the ability to move such varieties to the market. Recently, an open-access Cas enzyme derived from large language models (OpenCRISPR-1) was developed and shown to function effectively in human cells. In this study, we demonstrate the successful use of this nuclease in a polyploid plant species (Medicago sativa), with mono- or bi-allelic editing observed in 30% of genotypes bearing OpenCRISPR-1. These findings indicate that OpenCRISPR-1 holds promise to expand the use of gene editing technology in the breeding of polyploid crops.

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

In gene editing, CRISPR/Cas approaches are often limited by off-target effects. In in vivo approaches involving multiple cell types, off-targets may result from unintended targeting of the wrong cells. In this work, we propose a solution to this limitation by using a transcribed intron of the target gene as an endogenous trigger (intron triggers) for a novel conditional guide RNA (intcgRNA). In vitro, intcgRNAs were responsive to the presence of the trigger. As a proof-of-concept, the human IL2 receptor subunit gamma gene (IL2RG) was then targeted using both the intcgRNA and the corresponding conventional crRNA in two cell lines: the lymphocytic HPB-ALL cell line, where IL2RG is highly expressed, and the epithelial HeLa cell line, where it is not. Sanger sequencing revealed that the crRNA and intcgRNA Cas9 complexes edited IL2RG with similar efficiency in HPB-ALL, whereas only the crRNA edited IL2RG in HeLa. This shows that intcgRNA avoids targeting unwanted cells that do not express the target gene, which is particularly relevant for in vivo targeting. The triggers of choice for conditional guides have been microRNAs, but as short intronic RNAs are far more diverse, trigger introns could become biomarkers of cell identity that improve the precision of CRISPR-based manipulations in vivo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/714022v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ae60cdorg.highwire.dtl.DTLVardef@1556c03org.highwire.dtl.DTLVardef@1264a0dorg.highwire.dtl.DTLVardef@c7d47d_HPS_FORMAT_FIGEXP M_FIG C_FIG

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

We report the identification and functional validation of a 7SK RNA polymerase III promoter in the Mediterranean fruit fly, Ceratitis capitata. CRISPR/Cas9-based genetic control strategies for this global agricultural pest, including gene drives and precision guided sterile insect approaches, require efficient guide RNA expression, yet only a single U6 Pol III promoter had previously been validated for this purpose in C. capitata, and no 7SK promoter had been characterised in any Tephritid species. Using comparative genomics with Drosophila orthologues, we identified a previously unannotated 7SK gene in the C. capitata genome, confirmed its transcriptional activity by RT-PCR, and demonstrated that the cloned promoter drives functional guide RNA expression in CRISPR/Cas9-mediated knockouts of the white gene. Comparative analysis identified putative 7SK orthologues across the Tephritid fruit flies. The availability of this additional new Pol III promoter will enable multiplexed guide RNA strategies using distinct promoters, supporting more robust genetic control designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719894v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@156c203org.highwire.dtl.DTLVardef@db5eedorg.highwire.dtl.DTLVardef@353adforg.highwire.dtl.DTLVardef@ac079a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tracking Single Nucleotide Polymorphisms (SNPs) following CRISPR-Cas9 genome editing is a critical yet often labor-intensive step in modern genetic research. Although Sanger sequencing is the conventional method for definitive confirmation, it typically requires substantial time to generate results. In contrast, PCR-based restriction methods like CAPS (Cleaved Amplified Polymorphic Sequence) and dCAPS (derived CAPS) offer rapid and cost-effective alternatives. However, existing dCAPS primer design tools suffer from significant limitations and were largely developed for tracking polymorphisms in plant genomes. Concurrently, CRISPR-Cas9 gene editing requires strategies to prevent the re-cleavage of the edited allele, typically involving the modification of the Protospacer Adjacent Motif (PAM). To address these challenges, we developed EasyCAPS, a web-based tool that integrates dCAPS primer design with advanced functionalities for CRISPR experiments. EasyCAPS overcomes the shortcomings of previous software by enabling restriction enzyme pre-selection and optimizing designs for complex DNA sequences. Its key innovation is the "Hiding PAM" feature, which designs synonymous mutations to mask the Cas9 recognition site while accounting for codon usage bias, thereby facilitating one-step allelic exchange. The utility of the tool was demonstrated through practical applications targeting the HTA1, PHO84, and CAT5 genes, significantly accelerating both genotyping and gene editing processes. We conclude that EasyCAPS is an accessible solution that effectively streamlines molecular biology workflows.

Polygenic traits require the coordinated effects of multiple genes. Such complex traits have been a long-term target of study for geneticists, but multiplex CRISPR--the editing of multiple loci in the genome via multiple guide RNAs--is in its infancy. Reviewing 106 plant studies using multiplex CRISPR, we find that the multiplexing capacity has doubled every 5.4 years; furthermore, a systematic experiment with 8, 16, and 24 simultaneous targets in Arabidopsis thaliana reveals that efficiency of 24-plex editing can reach up to 73% across over one hundred third-generation transformed plants sequenced. Our experiment revealed that the level of multiplexing, or the number of the targets, causes minor efficiency reduction compared to the other uncontrolled factors such as gRNA design or variation across plants. When we model the decay in editing efficiency as a function of the gRNA number, actual efficiency is higher than the expectation from both Cas9 competition interference and simple joint editing stochasticity models. Rather, efficiency decayed with diminishing interference with more gRNAs with substantial overdispersion attributed to other efficiency factors, such as PAM identity. We predict that editing close to 100 genes in a plant can be feasible with reasonably large plant screens; however, feasible and reliable polygenic genome engineering will necessitate developments outside of [insert novelty of this study in how multiplex CRISPR was implemented, here]. Author ContributionsM.E.-A. conceived the project and secured funding. M.E.-A. and M.Es. designed the experimental strategy. M.Es. established the multiplex CRISPR and transformation pipelines in the laboratory, propagation through the T1 and T2 generations, and oversaw the first amplicon sequencing. Y.P. established the in-house iSeq amplicon sequencing protocol and contributed to cloning and genotyping pilots. M.Es supervised K.P. to construct cloning, bacterial transformations, plant growth, floral-dip transformations, and selection of T1 plants. E.K. contributed to early amplicon genotyping. J.K. propagated and sampled the T3 and J.K. and J.M. conducted the final amplicon sequencing panel. J.K. and M.E.-A. performed gene editing variant mapping, dataset quality control, and summarized results from published multiplex CRISPR studies. M.E.-A. modeled editing efficiency. J.K and M.E.-A. generated figures and wrote the first draft. All authors revised and improved the manuscript. J.K. and M.Es. contributed equally to this work and are designated as co-first authors.

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

The SCN1A gene encodes NaV1.1, a voltage-gated sodium channel protein that is necessary for neuronal excitability and whose loss-of-function mutations cause Dravet syndrome, a treatment-resistant childhood onset epilepsy. Gene replacement strategies for this syndrome are challenged by the large size of SCN1A and difficulty achieving stable cellular expression. Lentiviral vectors (LVVs) offer sufficient packaging capacity and genomic integration for defective SCN1A gene replacement. Here, we evaluated LVV-mediated delivery of different engineered SCN1A transgene sequences in human cells. LVV-transduced cells expressed full-length NaV1.1 protein that trafficked to the membrane and produced functional sodium currents. However, SCN1A transgene expression declined over time despite stable vector copy number, indicating post-integration regulatory limitations. Expression efficiency varied by SCN1A transgene sequence, with a codon-optimized variant showing higher expression despite lower LVV copy number. Treatment with sodium butyrate, a histone deacetylase inhibitor, significantly enhanced SCN1A transgene expression and partially rescued expression decay in a sequence-dependent manner. Incorporation of a ubiquitous chromatin opening element (UCOE) upstream of the promoter to maintain expression resulted in a trend of increased expression and increased responsiveness to butyrate. These findings demonstrate that sequence-specific and epigenetic factors may influence expression of large transgenes following lentiviral delivery, highlighting key challenges and design considerations for therapeutic SCN1A transgene expression.

Abstract/SummaryFIG4 deficiency is the cause of Charcot Marie Tooth type 4J, a neurological disorder characterized by enlarged lysosomes. Our CRISPR activation genome wide screen found that upregulation of PIKFYVE rescued the enlarged lysosome phenotype in cultured cells. To assess PIKFYVE upregulation treatment in vivo, we generated Fig4 deficient mice with CRISPR activation of Pikfyve in neurons. Pikfyve was increased 2 fold in whole brain of CRISPR activated mice. Pikfyve upregulation did not extend the 3 week survival of Fig4 deficient mice. Vacuolization of brain was not rescued. The data demonstrates that a 2 fold increase of Pikfyve is not sufficient to treat Fig4 deficiency. Further testing will be required to determine if a higher increase of Pikfyve can ameliorate the effects of FIG4 deficiency in vivo.

One feature key to the versatility of zebrafish as an animal model for biomedical research is the breadth of genetic tools available, including for transgenesis. While the Tol2 transposase system remains the gold standard, its efficiency can be highly variable. Here, we explored the potential of a complementary transgenesis system, Cp36, a large serine recombinase identified from Clostridium perfringens previously found to efficiently integrate target cargo into the human genome without a preinstalled attB site. We generated Cp36-based plasmid constructs for zebrafish transgenesis and compared their performance to matched Tol2 plasmids across multiple experimental contexts, including transient expression, germline transmission, and multi-transgene expression. Cp36 integrates small [~]3.5kb cargo into the zebrafish genome and transmits to the next generation as efficiently as Tol2, but Cp36 performance declines substantially for larger [~]7.5kb constructs. Both Cp36 and Tol2 have comparable efficiency in transiently expressing a second construct regardless of the transposase/recombinase used to integrate the first construct, indicating compatibility with sequential transgenesis strategies. In summary, we demonstrate that Cp36 functions as a new alternative transgenesis method in zebrafish.

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

Accurate prediction of CRISPR-Cas9 guide RNA (gRNA) editing efficiency remains limited, particularly outside human systems, where models trained on exogenous human datasets show poor generalization. We analyzed Cas9 efficiency and repair outcomes using novel endogenous editing data from four human cell types, two tomato cell types, and cells from giant river prawn and black soldier fly. While integrating publicly available predictors via ensemble frameworks improved performance, our analysis revealed hundreds of novel features affecting activity. Crucially, dominant features related to sites competition for gRNA, and local geometric properties varied across systems, highlighting the strong context dependence of Cas9 efficiency and arguing against a universal model. Interestingly, codon usage bias-based features also emerged as informative predictors, as they are proxies for chromatin accessibility. In contrast, trends in repair outcomes remained conserved. This work provides essential resources for more generalizable CRISPR guide design.

With the expansion of therapeutic gene editing technology, small animal models provide essential platforms to evaluate the function of these new approaches in vivo. As part of the Somatic Cell Genome Editing (SCGE) Consortium, we developed next-generation murine reporters that overcome current model limitations and broaden detectable in vivo editing outcomes. These include two mouse models built on the "traffic light" reporter concept. This system enables fluorescent detection of both gene repair (green) and CRISPR-generated indels (red) events following editing by a single guide and either dsDNA or single-stranded oligonucleotide donor. We also generated a third reporter model that efficiently detects A-base editor activity. Reporters were validated in cultured embryos, via germline editing, and through activation in vivo by AAV transduction or direct ribonucleoprotein delivery. Together, these new models provide a valuable resource for improved detection of genome editing events in vivo.

Polycomb repressive complexes PRC1 and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin production, a process whose reversal is a goal for the treatment of sickle cell disease and {beta}-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal hemoglobin (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-Cas9 mutagenesis screen identified a region in the EZH2 subunit where Cas9 induced exon 14 skipping (EZH2{Delta}14). EZH2{Delta}14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2{Delta}14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human {beta}-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting non-enzymatic domains within chromatin-modifying complexes. Key PointsO_LICRISPR-Cas9 screen across PRC1 and a saturating mutagenesis screen of PRC2 found the EZH2 CXC domain a desirable target for HbF induction C_LIO_LIthe EZH2-CXC domain leads to exon 14 exclusion, resulting in de-repression of HbF but maintenance of cell fitness. C_LI

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI