BGPT: Paper Review: CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium

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Paper reviewed: CRISPR/Cas9 multiplex editing in Dictyostelium

The core claim is that a transient, all-in-one CRISPR/Cas9 vector plus an endogenous Dictyostelium tRNA-processing sgRNA system enables efficient multiplex gene disruption, demonstrated on 5 PI3K genes with reported per-locus mutagenesis frequencies between 72.9% and 100%.

Long Explanation

Visual, skeptical paper analysis

Sekine & Kawata & Muramoto — “CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium” (Sci Rep, DOI: 10.1038/s41598-018-26756-z)

Quick scientific map (what they did → what they measured)

sgRNA expression strategy: compare U6 promoter vs tRNA-based Pol III processing; use cRT-PCR to validate mature sgRNA ends.
Cas9–sgRNA functional coupling: use dCas9-GFP localization changes as a proxy for sgRNA-driven complex formation.
Transient all-in-one editing: deliver Cas9+sgRNA from an episomal-like transient setup (temporary G418 selection) to avoid integrated drug cassette; test on tdTomato knock-in deletion and on endogenous acaA and pkaC.
Multiplex editing: build multiplex sgRNA vector (tRNA–sgRNA cassettes assembled via Golden Gate) to target 5 PI3K genes; report per-gene mutagenesis frequencies 72.9%–100% by mutation-detective PCR and confirm indels at all 5 loci by sequencing.

Evidence inventory (what is directly supported by measurements)

Claim in paper	Primary assay / metric	Reported value(s)	Strength (from given text)
tRNA processing yields higher sgRNA expression than U6	RT-PCR of sgRNA; cRT-PCR for mature ends	~10× higher; processed at tRNA-sgRNA junction	Strong (internal validation via end-mapping)
sgRNA expression supports Cas9 complex formation in nucleus	dCas9-GFP relocalization from nucleolus to nucleoplasm	Relocalization observed with sgRNA (qualitative + histogram SD)	Moderate (proxy, not direct complex quantification)
Transient Cas9+sgRNA causes tdTomato deletion efficiently	Single-cell fluorescence decrease + PCR confirmation	99.4% targeting efficiency (n=1618); ~700 bp deletion	Strong (orthogonal phenotype + PCR)
Transient multiplex targeting yields high indel rates at multiple loci	Mutation-detective PCR + sequencing at 5 loci	Per-gene mutagenesis 72.9%–100%; indels at all 5 loci in 4 clones	Strong-to-moderate (PCR screening + limited clone depth)

Skeptical critique: where the logic is strong vs where uncertainty remains

1) sgRNA expression: tRNA processing is plausible, and they validated ends

The study’s central engineering move is using the endogenous tRNA-processing machinery (RNase P/Z) to mature sgRNAs, validated by cRT-PCR showing cleavage precisely at the tRNA-sgRNA junction and termination in a Pol III terminator T-stretch.

2) Cas9 relocalization: useful proxy, but not a complete causal measurement

dCas9-GFP relocalization is consistent with higher sgRNA expression enabling formation/activation of Cas9–sgRNA complexes; however, localization changes are indirect. They rely on prior observations linking sgRNA levels to dCas9 nuclear relocalization.

3) Transient expression: reduced integrated cassette risk, but plasmid fragment retention is not eliminated

They argue transient all-in-one vector expression avoids genome integration of the drug cassette, and they note aggregation-negative clones lack Cas9-GFP signal—yet they explicitly state that retention of plasmid fragments cannot be eliminated.

4) Multiplex editing: strong on-target confirmation at 5 loci, but off-target analysis is limited in the provided text

The paper reports on-target indels at all five targeted PI3K loci across independent clones, consistent with efficient multiplex editing. However, the article’s discussion acknowledges CRISPR off-target indels can occur and proposes cost-based mitigation approaches (nickases, computational off-target tools), but in the provided text there is no deep, genome-wide off-target measurement.

5) Biological selection effects: small insertions may persist due to growth constraints

They observe that some indels introduce only 1–2 amino acids, and they propose these may be enriched because complete knockout of all five PI3K genes causes very poor growth in axenic medium; thus, growth conditions can bias which mutant genotypes survive.

Workflow diagram (faithful to the paper’s experimental design)

Evidence quality & limitations (what could change the conclusion)

Off-targets not comprehensively measured in the provided excerpt: they discuss known off-target mechanisms and mitigation (nickases, computational prediction), but genome-wide empirical off-target quantification is not evidenced in the text provided.
Mutation-detective PCR is a screening proxy: it indicates relative mutagenesis frequencies but does not replace locus-by-locus sequencing for every candidate allele. They do sequence selected clones, but the screening-to-confirmation mapping could be biased by PCR design and indel spectrum.
Selection effects from growth conditions: PI3K quintuple disruption causes poor axenic growth, so genotype survival can enrich specific indel types.
Transient-expression purity: they acknowledge plasmid fragment retention cannot be fully excluded. This matters for attributing phenotypes strictly to genome editing.

What would most strongly disprove or revise their main “efficient multiplex editing” claim?

Showing that the reported per-locus mutation-detective-PCR frequencies are not reproducible across independent lab replicates under similar transient conditions.
Empirical genome-wide off-target mapping revealing high rates of unexpected edits that could confound phenotypic interpretation—especially in multiplex settings.

Mechanistic context they rely on (with citations)

CRISPR/Cas9 cleavage depends on PAM presence (5′-NGG-3′ for SpCas9 in their background discussion) and mismatch sensitivity in a key upstream region (~12 nt upstream of PAM).
The paper motivates tRNA sgRNA expression by citing work showing sgRNA can be processed from tRNA structures.
For multiplex editing feasibility, they cite general multiplex CRISPR background (e.g., multiplex systems in other organisms).
For off-target mitigation concepts, they cite computational tools (COSMID, Cas-OFFinder) and nickase strategy.

Author reviews (BGPT)

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Updated: April 16, 2026