Discourse graph view — a rendering of record fields, no schema change

Alcalase pretreatment for CRISPR-Cas9 HDR knock-in in cell-walled Chlamydomonas reinhardtii (iced project)

Can Alcalase, an off-the-shelf serine endopeptidase, replace in-house autolysin to permeabilise the C. reinhardtii cell wall for Cas9 RNP plus HDR-donor electroporation and yield targeted knock-ins?

Source: Evaluation of Alcalase pretreatment for Chlamydomonas reinhardtii CRISPR knock-in (doi:10.57844/arcadia-pdu7-q2zz). Path record: arcadia-alcalase-path.json. Headline outcome abandoned. Alcalase digested walls but killed most cells before electroporation, gave near-background NAT resistance, and neither reagent yielded any on-target editing among 191 colonies screened; the project was iced (#TechnicalGap, #StrategicMisalignment). Abandonment reasons: technical-gap, strategic-misalignment. All graphs · Back to the pilot

How to read these graphs

A discourse graph breaks a piece of research into typed one-sentence nodes and draws how they bear on one another. The grammar is that of Discourse Graphs (Chan and Akamatsu). Read each graph left to right: a question, the experiments that addressed it, the results they produced, and the hypothesis every result is drawn against.

QUE Question: What the work set out to answer.
HYP Hypothesis: The answer the authors expected. Every result earns one edge against it.
EXP Experiment: One attempt at the question: a trial arm, an assay, a survey, an analysis.
RES Result: One finding from an experiment, with where in the source it comes from.
ISS Issue: An open question or untried branch the work leaves for someone else.

addresses: An experiment addresses the question.
produces: An experiment produces a result.
supports: A positive result that backs the hypothesis.
opposes: A negative result that counts as evidence against the hypothesis, or a refutation of an earlier claim.
informs: A partial or mixed result that bears on the hypothesis without settling it.
consistent with: A negative or inconclusive result that does not count either way: no positive control passed, or there is no sensitivity or power statement, so the absence of an effect cannot be told from a measurement that failed.
relation: A thin dotted line: a link the source itself states between two experiments, such as one being derived from another.

A dashed box marks a result the rule above counts as uninformative, or an issue. In the drawing, click a node to jump to its text below.

The graph

Read left to right. The question (QUE) is addressed by 6 experiments (EXP), each of which produces results (RES), and each result bears on the hypothesis (HYP) with one of four edges. 3 support it, 4 oppose it, 2 inform it, and 2 are merely consistent with it because the informativeness rule would not count them as evidence either way. Click a node to jump to its full text below.

supports positive finding
opposes informative negative, or refutation
informs partial finding
consistent with inconclusive or uninformative: no positive control passed, or no sensitivity statement
thin grey a relation the record itself states between two attempts
dashed box a result the informativeness rule marks uninformative, or an issue

The grammar is that of Discourse Graphs (Chan and Akamatsu): QUE, HYP, EXP, RES and ISS nodes with supports, opposes and informs edges. Every node and edge is derived from fields already in the Negative-Results Record; the edge a finding earns is decided by its outcomeClass and informativeness. Issues (ISS) hang below the graph as a strip: the open questions and untried branches the path leaves for someone else to pick up.

Nodes

Question and hypothesis

QUE: Can Alcalase, an off-the-shelf serine endopeptidase, replace in-house autolysin to permeabilise the C. reinhardtii cell wall for Cas9 RNP plus HDR-donor electroporation and yield targeted knock-ins?
HYP: Establish a robust, high-throughput-compatible CRISPR-Cas9 knock-in (HDR) system in cell-walled C. reinhardtii as a prerequisite for modular landing pads (Bigge et al. 2026, doi:10.57844/arcadia-61hk-a7gd); test Alcalase as an off-the-shelf substitute for autolysin.

Experiments (6 attempts)

Node	Attempt	Type	Headline	JSON
EXP	Flask- versus plate-grown starting cultures: no conclusion for knock-in; Alcalase survivors from flask tolerated electroporation better both formats yielded NAT-resistant colonies, but with no on-target events under either we can't say whether one format is better for knock-in	`wet-lab`	`inconclusive`	record
EXP	No on-target HDR insertion or indels detected at three loci among 191 NAT-resistant colonies (0/40 sequenced), under either pretreatment 0 of 191 colonies by PCR and 0 of 40 by sequencing showed on-target editing; delivery failure cannot be separated from editing or repair failure.	`wet-lab`	`inconclusive-no-positive-control`	record
EXP	Alcalase-treated CC-124 cells yield near-background NAT-resistant colonies after Cas9 RNP + donor electroporation; GeneArt-treated cells yield hundreds 0-16 CFU per plate on TAP + NAT; 6 of 8 treatments indistinguishable from the matched no-donor control; GeneArt 121-1,094	`wet-lab`	`negative-not-achievable`	record
EXP	Alcalase pretreatment (1:10, 1 h) cuts CC-124 viability at least ten-fold independent of electroporation 35 (flask) and 68 (plate) colonies after Alcalase vs > 1,200 after GeneArt; Alcalase survivors tolerated electroporation at 69% (flask) vs 38% (plate).	`wet-lab`	`negative-not-achievable`	record
EXP	Colony colour cannot serve as a visual screen for TLA knockouts under NAT selection Pale-green and white colonies appeared on all plates regardless of target; a third of white colonies re-greened off selection.	`wet-lab`	`negative-not-achievable`	record
EXP	Alcalase increases C. reinhardtii cell-wall permeability (OD-based leakage assay) Alcalase 22-23% leakage vs GeneArt 11% vs no enzyme 6-7% (n = 1 each).	`wet-lab`	`positive`	record

Results (11 findings)

Node	Finding	Id	Outcome and informativeness	Edge	JSON
RES	Do flask-grown (liquid, mid-log) and plate-grown (agar lawn) synchronised cultures differ in suitability for Alcalase/GeneArt pretreatment and knock-in? target: flask vs plate starting culture; electroporation survival, Alcalase-treated (electroporated/non-electroporated colony ratio) = 69% vs 38% (n = 1)	`culture-format` in arcadia-alcalase-culture-format	`inconclusive` uninformative no positive control; no sensitivity or power statement	consistent-with HYP	record
RES	Among NAT-resistant colonies from either pretreatment, did Cas9 RNP plus a 50 bp-homology-arm linear donor produce on-target HDR insertion or indels at CpSRP43, CpSRP54 or SRTA exon 1? target: on-target HDR knock-in at Cre04.g231026 / Cre11.g479750 / Cre10.g462200; colonies with ~2 kb HDR amplicon or sequence-confirmed edit = 0 (n = 191)	`hdr-ontarget` in arcadia-alcalase-hdr-knockin	`inconclusive-no-positive-control` uninformative no positive control	consistent-with HYP	record
RES	Does electroporation of Cas9 RNP plus linear NAT HDR donor into Alcalase-treated CC-124 cells yield NAT-resistant colonies above the no-donor background? target: Alcalase pretreatment + electroporation (RNP + NAT donor); NAT-resistant colonies per plate (TAP + 7.5 ug/mL NAT), 8 guide x culture conditions = 0-16 (n = 8)	`natr-alcalase` in arcadia-alcalase-transformation	`negative-not-achievable` informative internal positive arm passed; negative control clean; sensitivity or power stated	opposes HYP	record
RES	Does electroporation of Cas9 RNP plus linear NAT HDR donor into GeneArt-treated CC-124 cells yield NAT-resistant colonies above the no-donor background? target: GeneArt pretreatment + electroporation (RNP + NAT donor); NAT-resistant colonies per plate (TAP + 7.5 ug/mL NAT), 8 guide x culture conditions = 121-1094 (n = 8)	`natr-geneart` in arcadia-alcalase-transformation	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Do flask-grown CC-124 cells survive Alcalase pretreatment (1:10, 1 h) as well as GeneArt washing? target: Alcalase pretreatment (flask culture); colonies on TAP (100x dilution, no electroporation) = 35 (n = 1)	`viability-flask` in arcadia-alcalase-viability	`negative-not-achievable` informative internal positive arm passed; negative control clean; sensitivity or power stated	opposes HYP	record
RES	Do plate-grown CC-124 cells survive Alcalase pretreatment (1:10, 1 h) as well as GeneArt washing? target: Alcalase pretreatment (plate culture); colonies on TAP (100x dilution, no electroporation) = 68 (n = 1)	`viability-plate` in arcadia-alcalase-viability	`negative-not-achievable` informative internal positive arm passed; negative control clean; sensitivity or power stated	opposes HYP	record
RES	Can pale-green colony colour serve as a visual proxy for CpSRP43/CpSRP54 (TLA) knockouts among NAT-resistant colonies? target: colony colour as TLA-knockout readout; white colonies regaining green pigmentation off selection = 33 % (n = 96)	`visual-screen` in arcadia-alcalase-visual-screen	`negative-not-achievable` informative internal positive arm passed; negative control clean; sensitivity or power stated	opposes HYP	record
RES	Does Alcalase treatment permeabilise the cell wall of flask-grown CC-124 cells (detergent-induced pigment leakage)? target: Alcalase pretreatment (flask culture); OD662 released after Triton X-100 / OD662 before, percent = 23.36 % (n = 1)	`leak-alcalase-flask` in arcadia-alcalase-wall-permeability	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does GeneArt treatment permeabilise the cell wall of flask-grown CC-124 cells (detergent-induced pigment leakage)? target: GeneArt pretreatment (flask culture); OD662 released after Triton X-100 / OD662 before, percent = 11.21 % (n = 1)	`leak-geneart-flask` in arcadia-alcalase-wall-permeability	`partial` not-applicable rule applies to negative and inconclusive findings only	informs HYP	record
RES	Does Alcalase treatment permeabilise the cell wall of plate-grown CC-124 cells (detergent-induced pigment leakage)? target: Alcalase pretreatment (plate culture); OD662 released after Triton X-100 / OD662 before, percent = 21.84 % (n = 1)	`leak-alcalase-plate` in arcadia-alcalase-wall-permeability	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does GeneArt treatment permeabilise the cell wall of plate-grown CC-124 cells (detergent-induced pigment leakage)? target: GeneArt pretreatment (plate culture); OD662 released after Triton X-100 / OD662 before, percent = 10.92 % (n = 1)	`leak-geneart-plate` in arcadia-alcalase-wall-permeability	`partial` not-applicable rule applies to negative and inconclusive findings only	informs HYP	record

Relations between attempts

arcadia-alcalase-transformation wasDerivedFrom arcadia-alcalase-viability viability loss precedes and confounds delivery

Issues (6 open questions and untried branches)

ISS Has anyone gotten Alcalase pretreatment to work in C. reinhardtii? If so, what digestion time, concentration, and recovery conditions did you use? (open-question)
ISS If you've gotten autolysin preparations working consistently, which factors mattered most? (open-question)
ISS If we try making targeted knock-ins again, what 'need-to-haves' do you think we missed? (open-question)
ISS Develop a robust, CRISPR-compatible cell wall digestion method: improve autolysin (Shetty et al. 2025: mating at 28 C rather than 18 C) or gentler Alcalase (Walker et al. 2026, diatoms: osmotic support during and after digestion; titrate the enzyme dose down). (proposed-by-authors)
ISS Separate delivery failures from editing failures: restore a visual screen to confirm the guides, donor and selection work; include a donor-only, no-RNP control to estimate off-target NHEJ. (proposed-by-authors)
ISS Optimize on-target editing: co-target an endogenous selectable marker (Akella et al. 2021; Ross et al. 2024); tune RNP and donor concentrations and ratio (Kim et al. 2020 reached ~37% knock-in). (proposed-by-authors)