You are correct that gene editing in leukemia cell lines such as MOLM13 may be very different from ES cells regarding efficiency, clonal recovery, and overall robustness. The following are some observations and recommendations based on experience and the literature:

1. Feasibility of Endogenous Knock-in in MOLM13

Yes, it is possible, but much harder compared to ES cells because of:

Low homologous recombination (HR) efficiency. Sustained poor survival and recovery of single-cell clones. MOLM13 being more stressed-sensitive, including electroporation and antibiotic selection. There are key tips for its success. Some of which are discussed below:

A. Electroporation Strategy

Nucleofection is optimal for MOLM13; Lonza's 4D-Nucleofector system using the SF or SE buffer is optimal.

Utilize a high-fidelity Cas9 RNP complex (Cas9 protein + sgRNA) for improved editing efficiency and reduced toxicity.

Add HDR enhancer reagents, such as Alt-R HDR Enhencer (IDT) or SCR7, to enhance knock-in efficiencies.

B. Donor Template Design

Utilize ssDNA if your insert is smaller than 1 kb, or use AAV6 for >1 kb size templates.

Homology arms: starting with 500–800 bp each.

In the case of dTAG knock-in, require accurate in-frame fusion using the correct linkers (flexible GGGS repeats).

C. Selection and Cloning

Evade single-cell dilution with MOLM13 when strictly necessary.

Alternatively, use limiting dilution to small pools (e.g., 10–50 cells/well) or sort 2–3 cells/well.

Use conditioned media or co-culture with irradiated feeder cells (such as OP9) to facilitate growth.

Add IL-3 or SCF to media (some labs report advantage for MOLM13 viability).

D. Genotyping and Validation

Pre-screen by ddPCR, qPCR, or PCR with junction-specific primers.

Sequence-verify integration and off-targets.

dTAG system: confirm protein degradation with dTAG-13 or dTAGv-1 compound.

3. Exogenous Transgene to Safe Harbor

AAVS1 or CCR5 are general safe harbors, although less characterized in AML.

Lentiviral transduction is also a stable and convenient option for MOLM13 if precise integration is not necessary.

Consider inducible expression (e.g., Tet-On system) to achieve expression level of the endogenous locus.

4. Alternative Approach

If endogenous tagging is too inefficient, consider:

CRISPR interference (CRISPRi) to knockdown the endogenous gene.

Exogenous expression rescue with wildtype/mutant in parallel. This enables you to avoid clonal isolation but still test function.