Experiment: CRISPR knockout of CLEC12A in KMT2A-rearranged models, with and without a cooperating RAS lesion
also called CLEC12A KO KMT2A · Clec12a dependency test in KMT2A-r
Delete CLEC12A from KMT2A leukaemia cells and see whether they still grow, copying exactly what was done in a different leukaemia where deleting it worked. Do it with and without an extra RAS mutation, because the original experiment had one and we do not know whether that mattered.
The same thing, for a scientist
A direct replication of Mohanty 2025 [E1] in the index fusion family. **The design point that matters is the arm the original did not have**: a knockout in a fusion-only background, so that the NRAS-G12D confound is separated rather than inherited.
Design
Arms (four, plus controls).
- KMT2A::MLLT3 alone, CLEC12A knockout vs non-targeting sgRNA.
- KMT2A::MLLT3 + NRAS-G12D, knockout vs non-targeting.
- KMT2A::AFF1 (B-lineage) alone, knockout vs non-targeting - this is the arm that speaks to the index disease and the one most likely to be negative, since CLEC12A is rare on B-lineage blasts.
- NUP98::NSD1 + NRAS-G12D, knockout vs non-targeting - a positive control reproducing the published result in this lab's hands, without which a negative in arms 1-3 is uninterpretable.
Controls. Non-targeting sgRNA; a second independent CLEC12A sgRNA to exclude off-target; surface CLEC12A flow confirmation of knockout; and an essential-gene sgRNA to confirm the screen has dynamic range.
Readouts. Colony number in methylcellulose; annexin-V apoptosis; competitive growth over 21 days; and for the arms that survive that, engraftment and survival in NSG transplant.
The rule-67 analogue for a bench experiment: report the positive-control arm's effect size in the same figure as any negative arm. A flat knockout result is only a result if arm 4 moved.
Why the B-lineage arm is included despite an expected negative
CLEC12A is a myeloid antigen and is rare on B-lineage blasts [see gene-clec12a]. A negative in arm 3 is the expected outcome and is worth having anyway, because this project's index disease is infant KMT2A-rearranged B-ALL and there is currently no measurement of any kind - expression or dependency - for CLEC12A in it. A bounded negative in the index disease is more valuable to this graph than a third positive in AML.
| design | four-arm CRISPR knockout of CLEC12A across KMT2A::MLLT3, KMT2A::MLLT3+NRAS-G12D, KMT2A::AFF1 and a NUP98::NSD1+NRAS-G12D positive control, each versus non-targeting sgRNA and a second independent CLEC12A sgRNA |
|---|---|
| readout | methylcellulose colony number; annexin-V apoptosis; 21-day competitive growth; NSG engraftment and survival for arms with an in vitro phenotype |
| controls | non-targeting sgRNA; second independent sgRNA; flow confirmation of surface CLEC12A loss; essential-gene sgRNA for dynamic range; and the NUP98::NSD1 arm as an in-house positive control without which any negative is uninterpretable |
| cost tier | med |
| timeline | roughly 3 months to the in vitro read-out; 6-9 months including transplant arms |
- **A negative result here would be published nowhere**, so unless this lab records it in this graph the same bounded absence will be re-measured by the next reader.
- **Acute knockout and chronic antigen-low are different states.** A cell engineered to lose CLEC12A overnight is not the same as a clone selected over months to be CLL-1-low, and only the second is what patients relapse with.
- Which KMT2A-rearranged models actually express CLEC12A at baseline? The knockout is uninterpretable in a line that has none, so a flow survey must precede the experiment and may itself answer the question.
- Should a menin-inhibitor arm be included, so that the knockout is tested in the induced state the 2026 preprint describes rather than only at baseline?
- E1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
CRISPR/Cas9 depletion of Clec12a in NUP98::NSD1+NRAS-G12D-immortalised cells increased apoptosis and reduced colony numbers in vitro, and Clec12a deletion reduced leukaemic engraftment and prolonged survival in the murine model - establishing the protocol this experiment replicates.
Clec12a is required for the pathogenesis of NUP98::NSD1 AML. · Mohanty S, Charles Cano F, Gabdoulline R, Lai CK, Othman B, Sudarsanam H, et al. · Blood Adv 2025What this evidence cannot show
Abstract read in full from raw NCBI efetch XML on 2026-09-20; full text not read, so the exact sgRNA design, the delivery method and the colony-assay conditions are unknown to this entry and would have to be taken from the paper's methods before the replication is designed in detail.
Evidence that argues against this (1)
- If the published phenotype is NRAS-context-dependent, arm 4 of this experiment reproduces it and arms 1-3 do not, and the correct conclusion would be that the original result does not generalise - which is a useful outcome but not the one the hypothesis wants. Xiu Y et al. 2018
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
CLL-1-negative AML blasts persist and proliferate in patients after CLL-1-directed CAR T therapy has depleted the CLL-1-positive population, which is the clinical observation this experiment's fitness-cost prediction must explain.
Phase 1 Study of KITE-222, an Autologous CLL-1-Directed CAR T-cell Therapy in Patients with Relapsed/Refractory Acute Myeloid Leukemia. · Daver N, Blachly JS, Ghobadi A, Advani A, Muffly L, Garciaz S, et al. · Clin Cancer Res 2026What this evidence cannot show
Abstract read in full from raw NCBI efetch XML on 2026-09-20; full text not read. Adult R/R AML, n=12. Cited as the design constraint, not as a result about KMT2A.
Evidence that argues against this (1)
- If CLL-1-negative blasts are fully fit in patients, the knockout arms of this experiment should be negative, and a positive result would need explaining as a difference between acute genetic ablation and the chronic antigen-low state selected in a patient. Xiu Y et al. 2018
four-arm CRISPR knockout with an in-house NUP98::NSD1 positive control
- related to →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)
arm 3 is the B-lineage index-fusion arm, expected negative and worth having
- related to →KMT2A::MLLT3 (MLL-AF9), t(9;11)(p21.3;q23.3)
arms 1 and 2 are the myeloid index-fusion arms, with and without NRAS-G12D