Chapter Four · failure evidence
What CRISPR-Cas9 Gene Editing got wrong, from 76 dissertations
The records detail technical bottlenecks and experimental failures encountered when applying CRISPR-Cas9 genome editing across diverse biological systems. Investigators frequently faced severe cell death, ineffective delivery vectors, low homology-directed repair rates, and unpredictable guide activity. These records come from PhD theses at 20 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Nuclease expression, double-strand breaks, or essential gene disruption cause severe cytotoxicity and lethality
Cas9 endonuclease expression or double-strand break induction frequently triggered cell death, developmental arrest, or loss of pluripotency across model systems. These fatal consequences forced researchers to abandon standard knockout approaches in favor of knockdown or interference methods to maintain cell viability.
Tried and failed
homozygous gene knockout using CRISPR/Cas9 applied to human induced pluripotent stem cells. Reason: loss of target gene caused lethality and low cell viability
Investigating the role of sall4 during human cranial neural crest cells specification · Imperial
Tried and failed
CRISPR knock-in of degradation tag system applied to endogenous loci in difficult cell lines. Reason: low transfection efficiency and poor clone survival during selection
YAP and TAZ have Functionally Redundant Roles in Uveal Melanoma · MIT
Tried and failed
CRISPR-Cas9 endogenous fluorescent tagging of essential genes applied to pluripotency transcription factor endogenous locus. Reason: Off-target integration occurred, and biallelic targeting led to cell death or loss of pluripotency
The Regulation of Transcription Factors Binding Site Occupancies by Their Concentrations · EPFL
Considered and rejected
Considered and rejected: CRISPRi dCas9 knockdown in 786O cells abandoned for in vivo experiments due to dCas9-induced growth defects and outgrowth of unedited cells.
Pbaf-Associated Circadian Lncrna Adirf-As1 Regulates Renal Clear Cell Tumorigenesis · Penn
Considered and rejected
Considered and rejected: Rejected using CRISPR-Cas9 targeting alpha satellites to study centromeric DDR because generating tens of thousands of breaks per centromere exceeds physiological signaling thresholds.
Investigating the role of homologous recombination at human centromeres · Oxford
Tried and failed
CRISPR-Cas9 gene knockout applied to essential gene in cancer cell lines. Reason: Complete knockout resulted in cell lethality, preventing generation of stable clonal knockout lines.
Tried and failed
dual gRNA CRISPR-Cas9 knockout with antibiotic selection applied to cancer cell lines. Reason: specific gRNA pair co-transfections caused complete cell death post-transfection
The Role of RAD51AP1 in Drug Resistance in Glioblastoma Cells · Harvard
Tried and failed
CRISPR-Cas9 knockout of essential proteasome subunits applied to human cell line gene knockout. Reason: Knocking out the 5'-end of essential proteasomal catalytic subunits resulted in complete cell lethality.
Immunoproteasomes in microglial activation and inflammation · Imperial
Considered and rejected
Considered and rejected: Rejected using sgRNA-mediated CRISPR knockout of PNUTS in TREx-RTA-BCBL1 cells due to high cytotoxicity, adopting inducible shRNA knockdown instead.
The PNUTS-PP1 Complex Acts as an Intrinsic Barrier to Kaposi's Sarcoma Associated Herpesvirus Gene Expression and Replication · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Rejected CRISPR/Cas9 gene knockout in favor of shRNA knockdown because CRISPR requires mutating both alleles for complete knockout while shRNA provides efficient stable knockdown and avoids potential lethality of complete PDX1 loss.
Defining a Role for the Parahox Gene PDX1 in Colorectal Cancer · JScholarship
Tried and failed
CRISPR-Cas9 embryonic mosaic gene knockout applied to metabolic gene in non-model butterflies. Reason: Target gene disruption caused embryonic or larval lethality, preventing recovery of adult phenotypes.
The Role of Trehalase in Phenotypic Plasticity of the Buckeye Butterfly (Junonia coenia) · Cornell
Considered and rejected
Considered and rejected: CRISPR-Cas9-mediated gene editing rejected because Cas9 expression was toxic to Clostridium immunis.
The Host and Microbial Determinants of Activity by Commensal Clostridium immunis · DukeSpace
Tried and failed
heterologous catalytic dead CRISPR nuclease expression applied to bacterial host strain. Outcome: unstable. Reason: protein expression induced substantial host toxicity independent of guide RNA presence or selection
Considered and rejected
Considered and rejected: Abandoned UAS-Cas9 fat-body CRISPR knockout due to baseline Cas9-induced tissue toxicity under Cg-GAL4.
Considered and rejected
Considered and rejected: Rejected standard CRISPR knockout in favor of CRISPRi (dCas9-KRAB) for validating essential metabolic and UFMylation genes (UFSP2, G6PD) to avoid double-strand break lethality.
Investigating the cellular response to folate depletion · Harvard
Considered and rejected
Considered and rejected: Standard knockout-oriented CRISPR-Cas9 was rejected in favor of CRISPRi because complete ablation of essential epigenetic modifiers causes generic cell lethality rather than reflecting regulatory and environmental perturbations.
Considered and rejected
Considered and rejected: Rejected using CRISPR-Cas9 for high-MOI guide pooling in favor of CRISPRi with dCas9-KRAB to avoid cellular toxicity caused by excessive double-stranded breaks.
Investigating human disease mechanisms through population genetics and experimental genetic screens · Harvard
Considered and rejected
Considered and rejected: CRISPR-Cas9 knockout was rejected in favour of RNAi knockdown to avoid potential lethality and non-viable phenotypes from complete CBB cycle disruption
The significance of non-foliar photosynthesis on development and nutritional value in tomato fruit · University of Nottingham Repository
Tried and failed
CRISPR knock-in delivery via nucleofection applied to cancer cell lines. Reason: Severe nucleofection-induced cytotoxicity prevented survival and clonal expansion of stably edited cells.
Identifying the transcriptional drivers of dormancy in luminal A breast cancer · Imperial
Tried and failed
CRISPR-based transgenic strain generation via microinjection applied to mosquito population control target genes. Reason: Severe larval mortality prevented the establishment of viable transgenic lines across multiple microinjection attempts.
Considered and rejected
Considered and rejected: Rejected attempting CRISPR-Cas9 GRN R493X knockout in SH-SY5Y neuroblastoma cells due to complete cell death under antibiotic selection.
The Role of Progranulin in Frontotemporal Dementia · DSpace at UTSWMED
Viral and plasmid delivery systems suffer from low transduction efficiency compared to ribonucleoprotein complexes
Lentiviral, plasmid, and nanoparticle delivery strategies often yielded low transfection rates, oversized vector limits, or unedited target genomes. As a result, direct electroporation of Cas9 ribonucleoprotein complexes consistently outperformed viral or plasmid-based delivery methods.
Tried and failed
lentiviral CRISPR-Cas9 gene editing applied to hematopoietic stem and progenitor cells. Outcome: no signal. Reason: transduction occurred based on reporter expression but failed to produce detectable genomic DNA cleavage
Enhancing human NK cell development: from UCB-CD34+ cells to functional NK cells · Imperial
Lost to a baseline
Convective microfluidic transfection of large multicistronic CRISPR/Cas9 plasmids into iPSCs performed no better than standard Lipofectamine Stem (<1% efficiency).
Microfluidic Cell Processing for Personalized and Regenerative Medicine · Georgia Tech
Lost to a baseline
HDT-CRISPR-AuNP induced far lower gene editing and HDR integration frequencies than electroporated Cas9 RNP + HDT positive controls across Jurkats, T cells, and CD34+ cells.
Non-viral Gold Nanoparticle Mediated Delivery of CRISPR-Cas9 Ribonucleoprotein and Long DNA Transgenes into Primary Blood Cells · ResearchWorks
Lost to a baseline
Electroporation of 100 pmol free Cas9 RNP achieved 47.8 ± 15.3% editing in CD34+ HSPCs compared to 13.23 ± 0.12% for 3rd gen CRISPR-AuNP.
Development of Third-Generation Gold Nanoparticles (CRISPR-AuNP) for Enhanced CRISPR Delivery to Hematopoietic Stem and Progenitor Cells · ResearchWorks
Considered and rejected
Considered and rejected: Cas9-EGFP fusion protein was rejected due to much lower gene-editing efficiency in HSPCs/iPSCs compared to fluorophore-labeled CRISPR/Cas9-gRNA RNP complexes.
Establishment of a patient-specific iPSC model of stepwise leukemogenesis in severe congenital neutropenia · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Rejected lentiviral CRISPR/Cas9 delivery in CD34+ cells due to low editing efficiency and oversized 15.8 kb vector
Enhancing human NK cell development: from UCB-CD34+ cells to functional NK cells · Imperial
Considered and rejected
Considered and rejected: Rejected plasmid-based CRISPR-Cas9 (pSpCas9(BB)-2A-GFP) expression due to potential prolonged Cas9 toxicity/off-target effects and low transfection efficiency, switching to RNP delivery.
RUNDC1 Regulates Proinsulin/Insulin Trafficking and TGN Exit · ResearchWorks
Lost to a baseline
Electroporation of free Cas9 RNP achieved 76.5 ± 5.5% editing vs 52.7 ± 13.5% for electroporated 2nd gen CRISPR-AuNP.
Development of Third-Generation Gold Nanoparticles (CRISPR-AuNP) for Enhanced CRISPR Delivery to Hematopoietic Stem and Progenitor Cells · ResearchWorks
Tried and failed
all-in-one lentiviral delivery of Cas9 and gRNA applied to pooled genome-wide CRISPR knockout screen. Reason: Low viral titers resulted in poor infection efficiency requiring infeasibly high lentivirus volumes.
Discovery of cancer vulnerabilities in gene expression regulation pathway · Harvard
Tried and failed
dual viral vector CRISPR Cas9 gene editing applied to primary alveolar epithelial cells in vitro. Outcome: no signal. Reason: genomic DNA remained unedited across passages despite high vector expression efficiency
Considered and rejected
Considered and rejected: Rejected lentiviral delivery of CRISPR-Cas9-Cre due to genomic integration risks, gene silencing, and toxicity from permanent Cre expression, selecting adenoviral delivery instead.
Phosphorylierung der p53 H1-Helix: Rolle für Tumorsuppression und Tumorigenese p53 H1 helix phosphorylation: Role in tumor suppression and tumorigenesis · open_UMR Marburg DSpace 10.0
Considered and rejected
Considered and rejected: Rejected plasmid-based CRISPR Cas9 delivery to avoid risk of genomic integration and off-target effects, opting instead for Cas9 protein/crRNA delivery.
Targeting oncogenic microRNAs in malignant germ cell tumours. · Cambridge
Considered and rejected
Considered and rejected: Lentiviral delivery of CRISPR-Cas9 in intact primary mouse islets was rejected due to lack of editing success, leading to adoption of rat insulin promoter-driven shRNA vectors.
Unraveling The Beta Cell Translatome: Elucidation Of An Erk/hnrnpk/jund Axis · Penn
Considered and rejected
Considered and rejected: Rejected plasmid-based delivery of CRISPR-Cas9 constructs in favor of RNP complexes due to zero recovery of edited knock-in colonies.
Dissecting functional diversities of human sensory neuron subsets to develop a new humanized platform for pain therapy · JScholarship
Homology-directed repair and knock-in replacements exhibit poor integration efficiency
Targeted insertion of point mutations, reporter tags, and donor templates repeatedly failed to yield positive knock-in clones across hundreds of screened colonies. These homology-directed repair attempts were consistently impeded by non-functional repair pathways and an inability to achieve germline transmission in animal founders.
Tried and failed
single-vector CRISPR knock-in and knockout gene replacement applied to human embryonic stem cell lines. Reason: Target cells failed to replace wild-type alleles with the mutant knock-in construct.
Cell cycle remodelling during human embryonic stem cell differentiation · Imperial
Tried and failed
CRISPR/Cas9 homology-directed repair applied to human embryonic stem cell knock-in. Outcome: no signal. Reason: Zero of 192 sequenced clones showed successful incorporation of the target point mutation across three attempts
Tried and failed
CRISPR-Cas9 homology-directed repair with short donor templates applied to human B cell line gene editing. Outcome: no signal. Reason: extremely low or absent homology-directed repair efficiency yielding zero knock-in clones across hundreds screened
Investigating the effects of a novel ITP patient derived PRKDC mutation in a human B cell line. · Imperial
Tried and failed
CRISPR-Cas9 knock-in editing applied to candidate QTL variant validation. Reason: failed to generate edited mutants
Understanding complex behavior in C. elegans through natural variation · UT Austin
Considered and rejected
Considered and rejected: Homology-directed repair (HDR) via endonucleases (CRISPR/Cas9) at endogenous mutant loci in FA HSCs due to lack of functional homologous recombination machinery and low efficiency/error-prone nature of alternative repair pathways
Considered and rejected
Considered and rejected: Rejected direct CRISPR/Cas9 HDR mutagenesis in parental cells due to inefficient editing and p53 activation by double-strand breaks; adopted a multi-step LSL-knockin intermediate instead
Mutagenese des endogenen TP53-Lokus durch CRISPR/Cas9-vermittelte homologe Rekombination & Base Editing in Lungenkarzinomzellen Mutagenesis of the endogenous TP53-locus via CRISPR/Cas9-mediated homology-directed repair & Base Editing in lung cancer cells · open_UMR Marburg DSpace 10.0
Tried and failed
CRISPR Cas9 homology directed domain swap applied to murine zygote genome editing. Reason: Targeted knock-in failed to achieve germline transmission in founder animals
Tried and failed
CRISPR-Cas9 with single-stranded oligonucleotide knock-in applied to generating point mutation mouse founder lines. Reason: failed to yield viable knock-in founder lines across multiple loci
Modeling Autism Spectrum Disorder: Fragile X syndrome and Rett syndrome · MIT
Tried and failed
CRISPR-Cas9-mediated pronuclear microinjection of linearized plasmids applied to large transgene knock-in at safe-harbor loci. Outcome: no signal. Reason: Achieved zero successful genomic integration events in generated offspring despite using purified Cas9 protein.
Metabolic regulation of hierarchical decisions in the bone mesenchymal lineage · Imperial
Considered and rejected
Considered and rejected: Generating an inducible Mrgprb4Cre-ERT2 line via CRISPR/Cas9 was abandoned after multiple failed insertion attempts over two years.
A Critical Role For Mrgprb4 Touch Neurons In A Skin-Brain Pathway For Stress Resilience · Penn
Tried and failed
CRISPR/Cas9 N-terminal tag knock-in applied to endogenous gene tagging in Drosophila. Reason: Multiple tested gRNA combinations failed to yield successful integration
Cell-specific spindle alignment in epithelial tissue via RASSF proteins · Imperial
Tried and failed
CRISPR-Cas9 endogenous fluorescent tagging with degron insertion applied to endogenous gene tagging in nematodes. Reason: None
Investigating cohesin’s role in developmental gene regulation using the model organism C. elegans · Imperial
Engineered Cas variants and novel recruitment platforms underperform established baselines
Alternative Cas orthologs, PAM-flexible variants, and novel chimeric effectors repeatedly displayed weaker transcriptional repression or activation than established dCas9 baselines. Several engineered fusion platforms also lost gene silencing capability or showed undetectable activity compared to traditional regulatory recruitment tools.
Tried and failed
CRISPR activation targeting promoter with few sgRNAs applied to endogenous gene transcription in stem cells. Outcome: no signal. Reason: two promoter-targeting sgRNAs were insufficient to induce transcription compared to dense enhancer targeting
Lost to a baseline
dSaCas9 and dLbCas12a CRISPRoff yielded less efficient gene silencing than dSpCas9 CRISPRoff
Elucidating Gene Regulatory Roles of DNA Modifications and Active DNA Demethylation Pathways · ScholarlyCommons at Penn
Lost to a baseline
CRISPR RNAs with type I-E direct repeats transposed ~10^3-fold less efficiently in type I-F CASTs than native CAST crRNAs
Discovery of novel systems for genome editing · UT Austin
Lost to a baseline
Targeting the B2M TSS region with gRNA #231 using dCas9-KRAB yielded significant repression (0.69-fold), whereas novel CRISPR-GEMs showed weaker repression
Development and Application of Novel CRISPR-Based Epigenome Editors · DukeSpace
Lost to a baseline
PAM-flexible dCas9 variants (dxCas9-NG, dSpRY) exhibited weaker CRISPRi repression (5 to 30-fold) compared to wild-type dCas9 (~95-fold repression).
Synthetic CRISPR Tools for Information Processing in Bacteria · ResearchWorks
Lost to a baseline
dCas9-mediated CRISPRi targeting hisA failed to inhibit cell growth on minimal medium lacking histidine (cells remained viable), whereas Type IV-A1 CRISPRi completely suppressed growth (auxotrophy).
Characterization of Type IV-A1 CRISPR-interference on gene expression and plasmid replication · open_UMR Marburg DSpace 10.0
Lost to a baseline
CRISPR-Cas-mediated HMR recruitment showed undetectable Trp1 gene silencing, failing to reproduce the strong silencing growth defect achieved by Gal4DBD-Yif1 baseline.
CRISPR-Cas Tools for Engineering Genome Structure and Gene Expression · ResearchWorks
Lost to a baseline
At day 10 post-transduction, dCas9-KRAB achieved far stronger long-term repression of DKK1 (0.17-fold), IGFBPL1 (0.03-fold), and MLNR (0.23-fold) than any novel CRISPR-GEM (HDAC8, SIRT6, SETDB2)
Development and Application of Novel CRISPR-Based Epigenome Editors · DukeSpace
Lost to a baseline
Direct CRISPR-Cas fusions (dCas9-VP64 and dCpf1-VPR) were outperformed by RNA-mediated Com-VP64 recruitment in yeast reporter activation
Development of CRISPR-Cas tools towards rewiring 3D genome structure · ResearchWorks
Clonal selection suffers from off-target modifications and clone-to-clone variability
Constitutive Cas9 expression produced off-target DNA cleavage, uneven selection pressures, and divergent transcriptional drift across isolated single-cell clones. These artifacts caused inconsistent phenotypic behaviors among identically targeted cell lines, leading researchers to reject uncharacterized pools and constitutive expression systems.
Considered and rejected
Considered and rejected: Rejected classic single-colony CRISPR-Cas9 knockout for SNV modeling due to unpredictable karyotype-driven transcriptional drift and low (4%) editing efficiency.
Tried and failed
CRISPR knockout clone generation applied to tumorigenesis xenograft models. Outcome: did not generalise. Reason: different single-cell derived knockout clones showed discordant in vivo tumorigenic capacity despite identical targeting
SMARCB1 Maintains Lineage Fidelity in Clear Cell Renal Cell Carcinoma · Cambridge
Tried and failed
non-targeting sgRNA controls in pooled screens applied to pluripotent stem cell CRISPR screens. Reason: Non-targeting controls evade Cas9 double-strand break toxicity, causing inflated relative abundance compared to cut-inducing guides.
Controlling Cell Fate During Directed Differentiation of Human Beta Cells · Harvard
Considered and rejected
Considered and rejected: Rejected dCas9/CRISPRi-based sgRNA mismatch tuning library due to immunogenicity concerns and off-target risks from constitutive dCas9 expression
Considered and rejected
Considered and rejected: Rejected constitutive lentiCRISPR-v2 knockout system due to Cas9 off-target effects and clone-to-clone variability during clonal selection, switching to an inducible Tet-on iCas9 multiplex system
The role of the YTHDF m6A readers in neuroblastoma · IRIS - UNITN - prod
Considered and rejected
Considered and rejected: Rejected using constitutive CRISPR-Cas9 single-cell clonal lines due to clone-to-clone variability and long-term compensation artifacts, opting instead for Shield-1 inducible DD-Cas9.
Quantitative Analysis of Integrin Trafficking and Focal Adhesion Turnover to Study Integration of Early Endocytic Trafficking, Signaling and Migration · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Rejected using CRISPR-Cas9 for the primary whole-library genome-wide drop-out screen instead of shRNA, due to difficulty clonally expanding patient primary cells and risk of inducing compensatory changes.
TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Rejected the generation of CRISPR/Cas9 knockout clones directly from a heterogeneous pool of transduced cells without first isolating and validating a well-characterised single-cell Cas9-expressing clone.
Identifying and characterising new targets of inflammatory caspases · Imperial
Specific guide RNA designs show negligible cleavage activity and low on-target efficiency
Multiple candidate guide RNAs completely failed to generate detectable target cleavage at specific chromosomal loci. Even previously validated guide sequences or miniature Cas nucleases produced negligible mutation rates in experimental models.
Tried and failed
CRISPR-Cas9 targeted DNA cleavage using specific sgRNA applied to embryonic stem cell genome editing. Outcome: no signal. Reason: Specific guide RNA design exhibited low cleavage efficiency at the target chromosomal locus
Mechanism of recurrent DNA break clusters (RDCs) generation in mouse and human cells · Harvard
Tried and failed
CRISPR-Cas9 target cleavage applied to in vivo embryonic genomic DNA. Outcome: no signal. Reason: The designed guide RNA showed negligible cleavage activity resulting in an undetectable mutation rate.
Tried and failed
CRISPR-Cas9 locus deletion applied to cancer cell microRNA clusters. Outcome: no signal. Reason: poor cutting efficiency detected in mismatch cleavage assays
Targeting oncogenic microRNAs in malignant germ cell tumours. · Cambridge
Tried and failed
CRISPR-Cas9 disruption with published guide RNAs applied to transcription factor binding motif editing. Reason: previously published guide RNAs failed to yield viable edited cell models at the target locus
Transcripts and transcription at an estrogen regulated enhancer of CCND1 · Imperial
Tried and failed
engineered miniature CRISPR-Cas nucleases applied to reporter gene disruption in mammalian cells. Outcome: no signal. Reason: miniature Cas nucleases exhibited insufficient cleavage activity across varied nuclease-to-guide ratios
AAV TRANSDUCTION TRACING IN HEMATOPOIETIC CELLS BY TARGETED SCARRING OF GENOMIC SAFE HARBORS · Harvard
Considered and rejected
Considered and rejected: CRISPR-Cas9 knock-in of KRAS-G12V in ID8 cells using Kras guide 6 was abandoned due to low on-target guide efficiency
Targeting NAD+ metabolism in ovarian cancer · Imperial
Tried and failed
CRISPR-Cas9 targeted genome editing applied to single-nucleotide polymorphism introduction in yeast. Reason: guide RNAs targeting specific candidate loci were ineffective, requiring fallback to counterselectable marker-based mutagenesis
On Pleiotropy and Evolutionary Dynamics in the Budding Yeast Saccharomyces cerevisiae · Harvard
Inducible and split CRISPR designs exhibit leaky basal expression and poor regulatory control
Conditional and inducible editing architectures often showed persistent background activity in the absence of induction. This promoter leakiness created unintended genomic editing, high population heterogeneity, and severe host toxicity.
Tried and failed
split crRNA-tracrRNA inducible expression system applied to conditional CRISPR Cas9 genome editing. Reason: leaky basal expression caused unintended editing with no increase upon induction
Considered and rejected
Considered and rejected: Rejected tuning CRISPRi knockdown strength solely by titrating Cas9/gRNA inducer concentrations because it caused high cell-to-cell variability and population heterogeneity; adopted mismatched/truncated gRNA libraries instead.
Establishing the fast-growing bacterium Vibrio natriegens as a next-generation chassis for synthetic biology · open_UMR Marburg DSpace 10.0
Tried and failed
constitutive nuclease expression for genome editing applied to cyanobacterial CRISPR genome engineering. Outcome: worse than baseline. Reason: Constitutive Cas12a expression caused reduced transformation efficiency compared to inducible expression.
Engineering the membrane rotor ring of ATP synthases involved in photosynthesis · Imperial
Considered and rejected
Considered and rejected: Rejected constitutive two-plasmid CRISPRi system (dCas9 on single-copy, gRNA on multi-copy) due to severe growth inhibition on methanol
Engineering and evolving glyoxylate utilization in Ethylmalonyl-CoA Pathway-deficient Methylobacterium extorquens PA1 for the production of crotonate from methanol · open_UMR Marburg DSpace 10.0
Considered and rejected
Considered and rejected: Single-plasmid CRISPRi design with inducible dCas9 and constitutive gRNA expression due to persistent lethality on methanol in Δccr and ΔphaC strains from promoter leakiness
Engineering and evolving glyoxylate utilization in Ethylmalonyl-CoA Pathway-deficient Methylobacterium extorquens PA1 for the production of crotonate from methanol · open_UMR Marburg DSpace 10.0
Left open by the authors
Problems the authors named and did not get to.
Left open
Validate Cdh1 overexpression conclusions in human embryonic stem cells using CRISPR-mediated activation instead of the DHFR-TMP stabilization system. Blocker: Requires wet lab facilities, cell culture, and CRISPR genetic engineering apparatus.
Cell cycle remodelling during human embryonic stem cell differentiation · Imperial
Left open
Titrate Cas9 editing rates and optimize target site architecture to minimize multi-target deletions in the lineage tracing array. Blocker: Requires wet lab cell culture, CRISPR-Cas9 induction, and sequencing experiments
Left open
Generate chimeric CRISPR-Cas9 variants combining SpRY with hyperactive mutations and test cleavage efficiency and fidelity. Blocker: Requires a molecular biology wet lab to engineer, express, and assay Cas9 variants
Overcoming the limitations of CRISPR-based genome editing with kinetics-guided cryo-EM · UT Austin
Left open
Capture EBV episomes from spontaneous LCL lines exhibiting poor CRISPR/Cas9 editing and low proliferation. Blocker: Requires wet lab facilities, cell culture, CRISPR delivery, and biological samples (sLCL-B lines).
Investigating the germinal centre biology of an authentic Epstein-Barr virus strain · Imperial
Left open
Perturb candidate poised enhancers during the naïve-to-primed hESC transition using the optimized iCRISPRa system to assess gene activation effects. Blocker: Requires a wet lab, cell culture (hESCs), and experimental iCRISPRa assays.
Left open
Perform CRISPR-Cas9 loss-of-function mutations or overexpression of candidate genes to functionally validate identified chemoresistant transcriptomic signatures. Blocker: Requires a wet-lab environment, cell cultures, and CRISPR-Cas9 experimental assays.
Left open
Use CRISPR/Cas9 to create catalytically dead HDAC7 mutants to determine direct versus indirect deacetylation of targets and histones. Blocker: Requires wet-lab experiments, cell culturing, CRISPR/Cas9 genome editing, and biochemical assays.
Role Of Pre-Mrna Alternative Splicing In Protein Diversity And Epigenetics · Penn
Left open
Perform CRISPR-mediated enhancer screens across multi-gene GRBs or TADs during developmental transitions to validate predicted target and bystander gene responsiveness. Blocker: Requires wet lab experimentation with CRISPR/Cas9 systems in cell cultures during developmental transitions.
Responsiveness of genes to long-range transcriptional regulation · Imperial
Left open
Functionally validate candidate epigenetic regulators identified from the CRISPRi screen in primordial germ cell development models. Blocker: Requires a wet lab with mouse stem cell culture, genetic manipulation tools (CRISPRi), and differentiation assays
Left open
Use CRISPR/Cas9 endogenous engineering to insert primary GCAUG motifs into introns of secondary-motif-regulated candidates like Cd47 to test developmental timing impacts. Blocker: Requires a molecular biology wet lab and CRISPR/Cas9 cell engineering apparatus.
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