marchwarden/docs/stress-tests/M3.3-runs/08-comparative.log
Jeff Smith 13215d7ddb docs(stress-tests): M3.3 Phase A — calibration data collection
Issue #46 (Phase A only — Phase B human rating still pending, issue stays open).

Adds the data-collection half of the calibration milestone:

- scripts/calibration_runner.sh — runs 20 fixed balanced-depth queries
  across 4 categories (factual, comparative, contradiction-prone,
  scope-edge), 5 each, capturing per-run logs to docs/stress-tests/M3.3-runs/.
- scripts/calibration_collect.py — loads every persisted ResearchResult
  under ~/.marchwarden/traces/*.result.json and emits a markdown rating
  worksheet with one row per run. Recovers question text from each
  trace's start event and category from the run-log filename.
- docs/stress-tests/M3.3-rating-worksheet.md — 22 runs (20 calibration
  + caffeine smoke + M3.2 multi-axis), with empty actual_rating columns
  for the human-in-the-loop scoring step.
- docs/stress-tests/M3.3-runs/*.log — runtime logs from the calibration
  runner, kept as provenance. Gitignore updated with an exception
  carving stress-test logs out of the global *.log ignore.

Note: M3.1's 4 runs predate #54 (full result persistence) and so are
unrecoverable to the worksheet — only post-#54 runs have a result.json
sibling. 22 rateable runs is still within the milestone target of 20–30.

Phases B (human rating) and C (analysis + rubric + wiki update) follow
in a later session. This issue stays open until both are done.

Co-Authored-By: Claude Opus 4.6 (1M context) <noreply@anthropic.com>
2026-04-08 20:21:47 -06:00

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Researching: Compare CRISPR-Cas9 and CRISPR-Cas12 for in vivo gene editing.
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╭─────────────────────────────────── Answer ───────────────────────────────────╮
│ CRISPR-Cas9 and CRISPR-Cas12a (formerly Cpf1) are both widely used │
│ RNA-guided nucleases adapted for genome editing, including in vivo │
│ applications, but they differ meaningfully in mechanism, structure, PAM │
│ requirements, cutting pattern, guide RNA architecture, specificity, and │
│ practical suitability for in vivo delivery. │
│ │
│ **Mechanism and DNA Cleavage:** Cas9 (most commonly from Streptococcus │
│ pyogenes, SpCas9) cleaves both DNA strands at the same position, producing │
│ blunt-ended double-strand breaks (DSBs) [Source 7]. Cas12a, by contrast, │
│ introduces staggered cuts that leave 45 nucleotide 5 overhangs [Sources 2, │
│ 7]. These sticky ends generated by Cas12a may enhance homology-directed │
│ repair (HDR) efficiency compared to Cas9's blunt ends [Source 2]. │
│ │
│ **PAM Sequence:** Cas9 requires an NGG PAM (protospacer adjacent motif) on │
│ the non-template strand downstream of the target; Cas12a recognizes a T-rich │
│ PAM (typically TTTV) upstream of the target on the non-template strand │
│ [Sources 2, 7]. This difference expands the targeting range of Cas12a to │
│ AT-rich genomic regions where Cas9 is limited. │
│ │
│ **Guide RNA:** Cas9 uses a two-component guide (crRNA + tracrRNA, often │
│ fused as sgRNA), while Cas12a requires only a single crRNA with a short │
│ direct repeat and processes its own pre-crRNA array, enabling multiplexed │
│ editing from a single transcript [Sources 2, 7, 13]. │
│ │
│ **Specificity and Off-Target Effects:** Kinetic studies show Cas12a exhibits │
│ greater target specificity than Cas9, attributed to a more stringent DNA │
│ unwinding mechanism that requires more extensive complementarity before │
│ cleavage [Source 5]. Cas12a tolerates fewer mismatches between the guide RNA │
│ and target, resulting in fewer off-target cuts [Sources 2, 5]. │
│ │
│ **Editing Efficiency:** In comparative studies using ribonucleoprotein (RNP) │
│ delivery in rice (OsPDS gene), Cas9 and Cas12a showed different efficiencies │
│ depending on the target site [Source 1]. In Chlamydomonas reinhardtii, both │
│ Cas9 and Cas12a RNPs co-delivered with ssODN repair templates achieved │
│ similar total editing levels of 2030% [Source 4]. Context and target site │
│ selection significantly influence which enzyme performs better. │
│ │
│ **In Vivo Delivery Considerations:** Both enzymes can be delivered via AAV │
│ vectors, lipid nanoparticles (LNPs), or as RNPs via electroporation [Sources │
│ 21, 24]. A critical practical consideration is size: SpCas9 (~4.2 kb coding │
│ sequence) is near the AAV packaging limit (~4.74.8 kb), leaving little room │
│ for promoter and regulatory elements [Sources 20, 21]. Cas12a variants │
│ (including engineered compact forms such as EbCas12a) can be packaged │
│ together with their crRNA within a single AAV vector, which is a significant │
│ advantage for in vivo delivery [Sources 19, 20, 21]. A miniature Cas12f1 │
│ variant has also demonstrated efficacy for in vivo retinal gene therapy │
│ [Source 12]. │
│ │
│ **Clinical and Therapeutic Status:** CRISPR-Cas9 is currently the dominant │
│ nuclease in clinical trials for both ex vivo and in vivo genome editing │
│ [Sources 8, 11]. Cas12a is gaining traction in therapeutic research, │
│ particularly where higher specificity or AAV-compatible delivery is required │
│ [Sources 9, 13, 22]. │
│ │
│ **Summary Table:** │
│ - DNA cut type: Cas9 = blunt; Cas12a = staggered (5 overhang) │
│ - PAM: Cas9 = NGG (3); Cas12a = TTTV (5) │
│ - Guide RNA: Cas9 = sgRNA (crRNA+tracrRNA); Cas12a = crRNA only │
│ - Multiplexing: Cas9 = limited; Cas12a = inherent crRNA array processing │
│ - Specificity: Cas12a generally higher │
│ - AAV compatibility: Cas12a variants better suited │
│ - Clinical use: Cas9 more established; Cas12a emerging │
╰──────────────────────────────────────────────────────────────────────────────╯
Citations
┏━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━┓
┃ # ┃ Title / Locator ┃ Excerpt ┃ Conf ┃
┡━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━┩
│ 1 │ What's the Difference Between │ Cas9...cleaves both strands of │ 0.95 │
│ │ Cas9 and Cas12a Nucleases? | │ DNA at the same point. This │ │
│ │ The Scientist │ creates a blunt end │ │
│ │ https://www.the-scientist.com │ double-stranded break (DSB)... │ │
│ │ /what-s-the-difference-betwee │ For Cas9 to function, the │ │
│ │ n-cas9-and-cas12a-nucleases-7 │ protospacer adjacent motif │ │
│ │ 2481 │ (PAM)—a two to six base pair │ │
│ │ │ sequence—NGG...must sit │ │
│ │ │ immediately downstream of the │ │
│ │ │ target on the opposite strand. │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 2 │ Cas9 versus Cas12a/Cpf1: │ Cas9 and Cas12a have distinct │ 0.97 │
│ │ Structure-function │ evolutionary origins and │ │
│ │ comparisons and implications │ exhibit different structural │ │
│ │ for genome editing - PubMed │ architectures, resulting in │ │
│ │ https://pubmed.ncbi.nlm.nih.g │ distinct molecular │ │
│ │ ov/29790280/ │ mechanisms... We discuss │ │
│ │ │ implications for genome │ │
│ │ │ editing, and how they may │ │
│ │ │ influence the choice of Cas9 │ │
│ │ │ or Cas12a for specific │ │
│ │ │ applications. │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 3 │ CRISPR-Cas12a More Precise │ Cas12a...is, according to │ 0.90 │
│ │ Than CRISPR-Cas9 │ scientists at the University │ │
│ │ https://www.genengnews.com/to │ of Texas at Austin │ │
│ │ pics/genome-editing/crispr-ca │ (UT-Austin), more effective │ │
│ │ s12a-more-precise-than-crispr │ and precise... Because Cas │ │
│ │ -cas9/ │ enzymes occasionally fail to │ │
│ │ │ cut DNA in the right places, │ │
│ │ │ or even cut at all, they worry │ │
│ │ │ developers, who want to modify │ │
│ │ │ genomes with surgical │ │
│ │ │ precision, especially in │ │
│ │ │ therapeutic applications. │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 4 │ Comparison of CRISPR/Cas9 and │ We found that Cas9 and Cas12a │ 0.92 │
│ │ Cas12a for gene editing in │ RNPs- co-delivered with ssODN │ │
│ │ Chlamydomonas reinhardtii - │ repair templates- induced │ │
│ │ ScienceDirect │ similar levels of total │ │
│ │ https://www.sciencedirect.com │ editing, achieving as much as │ │
│ │ /science/article/pii/S2211926 │ 2030 % in all │ │
│ │ 424004089 │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 5 │ Comparison of │ Comparison of │ 0.88 │
│ │ CRISPR-Cas9/Cas12a │ CRISPR-Cas9/Cas12a │ │
│ │ Ribonucleoprotein Complexes │ Ribonucleoprotein Complexes │ │
│ │ for Genome Editing Efficiency │ for Genome Editing Efficiency │ │
│ │ in the Rice Phytoene │ in the Rice Phytoene │ │
│ │ Desaturase (OsPDS) Gene - PMC │ Desaturase (OsPDS) Gene │ │
│ │ https://pmc.ncbi.nlm.nih.gov/ │ │ │
│ │ articles/PMC6973557/ │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 6 │ Current and Prospective │ Current and Prospective │ 0.87 │
│ │ Applications of CRISPR-Cas12a │ Applications of CRISPR-Cas12a │ │
│ │ in Pluricellular Organisms - │ in Pluricellular Organisms... │ │
│ │ PMC │ Mol Biotechnol. 2022 Aug │ │
│ │ https://pmc.ncbi.nlm.nih.gov/ │ 8;65(2):196205. doi: │ │
│ │ articles/PMC9841005/ │ 10.1007/s12033-022-00538-5 │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 7 │ When size matters: A novel │ When size matters: A novel │ 0.90 │
│ │ compact Cas12a variant for in │ compact Cas12a variant for in │ │
│ │ vivo genome editing - PMC │ vivo genome editing │ │
│ │ https://pmc.ncbi.nlm.nih.gov/ │ │ │
│ │ articles/PMC11253977/ │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 8 │ When size matters: A novel │ Altogether, the components of │ 0.91 │
│ │ compact Cas12a variant for in │ the EbCas12a system are well │ │
│ │ vivo genome editing - │ below the 4.8-kb packaging │ │
│ │ ResearchGate │ limit of AAVs, enabling │ │
│ │ https://www.researchgate.net/ │ successful packaging in the │ │
│ │ publication/382328745_When_si │ AAV9 │ │
│ │ ze_matters_A_novel_compact_Ca │ │ │
│ │ s12a_variant_for_in_vivo_geno │ │ │
│ │ me_editing │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 9 │ Therapeutic In Vivo Gene │ our current results prove that │ 0.88 │
│ │ Editing Achieved by a │ the miniature Cas12f1 system │ │
│ │ Hypercompact CRISPR System - │ is a promising gene editing │ │
│ │ Advanced Science │ tool for retinal gene therapy │ │
│ │ https://advanced.onlinelibrar │ │ │
│ │ y.wiley.com/doi/10.1002/advs. │ │ │
│ │ 202308095 │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 10 │ Delivery of CRISPR-Cas tools │ AAV is one of the most │ 0.90 │
│ │ for in vivo genome editing │ commonly used vector systems │ │
│ │ therapy: Trends and │ to date, but immunogenicity │ │
│ │ challenges - ScienceDirect │ against capsid, liver toxicity │ │
│ │ https://www.sciencedirect.com │ at high dose, and potential │ │
│ │ /science/article/pii/S0168365 │ genotoxicity caused by │ │
│ │ 92200027X │ off-target mutagenesis and │ │
│ │ │ genomic integration remain │ │
│ │ │ unsolved. │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 11 │ CRISPR-Based Therapeutic │ These Cas proteins are more │ 0.87 │
│ │ Genome Editing - DSpace@MIT │ compatible with AAV delivery, │ │
│ │ https://dspace.mit.edu/bitstr │ enabling additional vector │ │
│ │ eam/handle/1721.1/138388.2/ni │ design options such as │ │
│ │ hms-1576523.pdf?sequence=4&is │ expanded promoter choices and │ │
│ │ Allowed=y │ a streamlined delivery. │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 12 │ Revolutionizing in vivo │ Genome editing using the │ 0.85 │
│ │ therapy with CRISPR/Cas │ CRISPR/Cas system has │ │
│ │ genome editing: │ revolutionized the field of │ │
│ │ breakthroughs, opportunities │ genetic engineering, offering │ │
│ │ and challenges - Frontiers │ unprecedented opportunities │ │
│ │ https://www.frontiersin.org/j │ for therapeutic applications │ │
│ │ ournals/genome-editing/articl │ in vivo. │ │
│ │ es/10.3389/fgeed.2024.1342193 │ │ │
│ │ /full │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 13 │ CRISPR Clinical Trials: A │ CRISPR Clinical Trials: A 2024 │ 0.80 │
│ │ 2024 Update - Innovative │ Update - Innovative Genomics │ │
│ │ Genomics Institute │ Institute (IGI) │ │
│ │ https://innovativegenomics.or │ │ │
│ │ g/news/crispr-clinical-trials │ │ │
│ │ -2024/ │ │ │
├─────┼───────────────────────────────┼────────────────────────────────┼───────┤
│ 14 │ Alt-R CRISPR-Cas9 vs Cas12a │ The two most popular enzymes │ 0.83 │
│ │ systems | IDT │ used in CRISPR genome editing │ │
│ │ https://www.idtdna.com/pages/ │ are Cas9 and Cas12a (Cpf1). │ │
│ │ technology/crispr/crispr-geno │ These enzymes are highly │ │
│ │ me-editing/Alt-R-systems │ functional, do not require │ │
│ │ │ binding to other enzymes as is │ │
│ │ │ the case for type I CRISPR │ │
│ │ │ systems, and can be readily │ │
│ │ │ programmed to target the │ │
│ │ │ desired genomic DNA site. │ │
└─────┴───────────────────────────────┴────────────────────────────────┴───────┘
Gaps
┏━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓
┃ Category ┃ Topic ┃ Detail ┃
┡━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┩
│ source_not_found │ Head-to-head in vivo │ Most comparative studies │
│ │ efficacy data in mammals │ focused on plants (rice) or │
│ │ across multiple tissue │ algae (Chlamydomonas) or │
│ │ types │ used in vitro/ex vivo │
│ │ │ models. Rigorous │
│ │ │ side-by-side in vivo │
│ │ │ mammalian comparisons of │
│ │ │ Cas9 vs. Cas12a across │
│ │ │ liver, muscle, CNS, and eye │
│ │ │ were not identified in │
│ │ │ available sources. │
├──────────────────┼─────────────────────────────┼─────────────────────────────┤
│ source_not_found │ Immunogenicity comparison │ While immunogenicity of │
│ │ between Cas9 and Cas12a in │ Cas9 is well-documented as │
│ │ vivo │ a challenge for in vivo │
│ │ │ delivery, direct │
│ │ │ comparative immunogenicity │
│ │ │ data for Cas12a in humans │
│ │ │ or animal models was not │
│ │ │ available in the gathered │
│ │ │ sources. │
├──────────────────┼─────────────────────────────┼─────────────────────────────┤
│ source_not_found │ Cas12a clinical trial data │ The IGI clinical trials │
│ │ │ update and other sources │
│ │ │ confirm Cas9 dominance in │
│ │ │ trials but do not provide │
│ │ │ details on approved or │
│ │ │ ongoing Cas12a-specific │
│ │ │ clinical trials. │
├──────────────────┼─────────────────────────────┼─────────────────────────────┤
│ source_not_found │ Detailed off-target │ While Cas12a is reported to │
│ │ profiling comparison in │ be more specific than Cas9 │
│ │ vivo │ based on kinetic studies, │
│ │ │ comprehensive in vivo │
│ │ │ off-target profiling │
│ │ │ comparing both enzymes │
│ │ │ systematically across the │
│ │ │ same targets was not │
│ │ │ available in the sources. │
└──────────────────┴─────────────────────────────┴─────────────────────────────┘
Discovery Events
┏━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━┓
┃ ┃ Suggested ┃ ┃ ┃
┃ Type ┃ Researcher ┃ Query ┃ Reason ┃
┡━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━┩
│ related_research │ arxiv │ Cas12a vs Cas9 in │ Head-to-head in │
│ │ │ vivo editing │ vivo mammalian │
│ │ │ efficiency │ comparisons are a │
│ │ │ off-target │ critical gap; │
│ │ │ mammalian │ preprint servers │
│ │ │ therapeutic │ may have more │
│ │ │ comparison 2023 │ recent │
│ │ │ 2024 │ unpublished data │
├──────────────────┼───────────────────┼───────────────────┼───────────────────┤
│ related_research │ database │ CRISPR Cas12a │ Clinical adoption │
│ │ │ clinical trials │ of Cas12a in vivo │
│ │ │ ClinicalTrials.go │ is poorly │
│ │ │ v 2023 2024 │ characterized; a │
│ │ │ │ ClinicalTrials.go │
│ │ │ │ v database search │
│ │ │ │ would clarify │
│ │ │ │ current status │
├──────────────────┼───────────────────┼───────────────────┼───────────────────┤
│ related_research │ arxiv │ Cas12a │ Immunogenicity is │
│ │ │ immunogenicity │ a key barrier for │
│ │ │ pre-existing │ in vivo Cas9 │
│ │ │ immunity in vivo │ delivery; whether │
│ │ │ gene therapy │ Cas12a poses │
│ │ │ human │ fewer immune │
│ │ │ │ challenges is │
│ │ │ │ clinically │
│ │ │ │ important but not │
│ │ │ │ covered in │
│ │ │ │ sources │
├──────────────────┼───────────────────┼───────────────────┼───────────────────┤
│ new_source │ database │ compact Cas12a │ Compact Cas12a │
│ │ │ EbCas12a AsCas12a │ variants show │
│ │ │ in vivo liver │ promise for AAV │
│ │ │ lung CNS │ delivery; recent │
│ │ │ therapeutic │ therapeutic in │
│ │ │ editing 2024 │ vivo data would │
│ │ │ │ strengthen the │
│ │ │ │ comparison │
└──────────────────┴───────────────────┴───────────────────┴───────────────────┘
Open Questions
┏━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓
┃ Priority ┃ Question ┃ Context ┃
┡━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┩
│ high │ Does Cas12a's staggered cutting │ Sources note that staggered │
│ │ pattern result in meaningfully │ cuts may enhance HDR, but │
│ │ higher HDR rates than Cas9's │ comparative in vivo HDR │
│ │ blunt cuts in vivo in │ efficiency data in mammals was │
│ │ therapeutically relevant cell │ not found in the gathered │
│ │ types? │ evidence. │
├──────────┼─────────────────────────────────┼─────────────────────────────────┤
│ high │ Are there pre-existing │ Immunogenicity is a known │
│ │ antibodies or T-cell responses │ challenge for Cas9 in vivo; │
│ │ against Cas12a proteins in │ whether Cas12a, being from │
│ │ humans that would limit its │ different bacterial origins, │
│ │ therapeutic use, as has been │ faces similar or lesser immune │
│ │ documented for SpCas9? │ barriers in human patients is │
│ │ │ clinically critical. │
├──────────┼─────────────────────────────────┼─────────────────────────────────┤
│ high │ Can compact Cas12a variants │ Compact variants fit within AAV │
│ │ (e.g., EbCas12a, Cas12f) │ packaging limits better than │
│ │ consistently match or exceed │ Cas9, but their in vivo editing │
│ │ SpCas9 editing efficiency in │ efficiency relative to SpCas9 │
│ │ vivo across diverse tissue │ across tissues such as liver, │
│ │ types? │ muscle, and CNS needs │
│ │ │ systematic evaluation. │
├──────────┼─────────────────────────────────┼─────────────────────────────────┤
│ medium │ How does Cas12a's inherent │ Cas12a can process its own │
│ │ crRNA array processing and │ pre-crRNA array, enabling │
│ │ multiplexing capability │ multiplexed targeting from a │
│ │ translate to in vivo │ single transcript, which is │
│ │ combinatorial therapeutic │ noted as an advantage but its │
│ │ strategies compared to │ in vivo therapeutic │
│ │ Cas9-based multiplex │ exploitation is not │
│ │ approaches? │ well-characterized in available │
│ │ │ sources. │
├──────────┼─────────────────────────────────┼─────────────────────────────────┤
│ medium │ What is the current status of │ The 2024 CRISPR clinical trials │
│ │ Cas12a-specific clinical trials │ update from IGI and Frontiers │
│ │ for in vivo gene therapy, and │ review both highlight Cas9 │
│ │ how do their safety profiles │ dominance in clinical trials, │
│ │ compare to Cas9-based trials? │ but Cas12a clinical translation │
│ │ │ remains poorly documented. │
└──────────┴─────────────────────────────────┴─────────────────────────────────┘
╭───────────────────────────────── Confidence ─────────────────────────────────╮
│ Overall: 0.82 │
│ Corroborating sources: 14 │
│ Source authority: high │
│ Contradiction detected: False │
│ Query specificity match: 0.85 │
│ Budget status: spent │
│ Recency: current │
╰──────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────── Cost ────────────────────────────────────╮
│ Tokens: 54153 │
│ Iterations: 3 │
│ Wall time: 117.16s │
│ Model: claude-sonnet-4-6 │
╰──────────────────────────────────────────────────────────────────────────────╯
trace_id: 9e436db7-fcde-4d0f-a568-c468ae4d419c