Section 1: Modelling Patient Variants Using CRISPR-Cas9 in C. elegans

What is a Variant of Uncertain Significance (VUS)?

Mostly missense variants that cannot be classified as pathogenic or benign, making them not clinically actionable.

What percentage of missense variants in ClinVar are VUS?

~58% of 207,000+ reported missense variants.

What is the clinical impact of a VUS designation?

Patients cannot join gene therapy trials or receive targeted treatments; it is a dead end for clinical action.

How does C. elegans help solve the VUS challenge?

It provides functional evidence to reclassify VUS as "likely pathogenic" or "likely benign," bridging genomic sequencing to clinical diagnosis.

How fast can new worm strains with patient mutations be generated using CRISPR-Cas9?

In 2–3 weeks.

What are the two requirements for successfully modelling a human VUS in C. elegans?

① Conservation of the mutated residue at the orthologous position; ② Shared biological function between species.

What disease was modelled in the Lange et al. (2021) case study?

Joubert Syndrome (JBTS), a ciliopathy with "molar tooth sign" brain malformation.

What were the two patient mutations in human B9D2 studied by Lange et al.?

P74S (in the B9 domain) and G155S (in the C-terminal region).

What is the worm orthologue of human B9D2?

mksr-2 (encoding MKSR-2 protein), with 63.4% similarity to human B9D2.

Why did Lange et al. use endogenous tagging with mNeonGreen?

To avoid overexpression artefacts by knocking the tag in at the 5' end of the endogenous mksr-2 locus.

What genotype was created to mimic the patient's biallelic state?

Compound heterozygotes (P74S/G155S), plus carrier controls (P74S/+, G155S/+).

What did super-resolution microscopy reveal about the two variants?

G155S caused severe reduction and disorganized Transition Zone (TZ) distribution; P74S caused a milder reduction.

Why did the study use an nphp-4(Δ) sensitized background?

Because single TZ mutants often appear normal; the sensitized background makes ciliary structure defects visible.

What behavioural assays were used to assess variant severity?

Roaming (food foraging) and osmotic avoidance (high osmolarity barriers).

Which variant showed a stronger defect in osmotic sensing?

G155S.

How was ciliary gating (barrier integrity) tested?

By testing for leakage of proteins normally excluded from the cilium (RPI-2, TRAM-1) across the transition zone.

What was the gating result for P74S vs. G155S?

G155S disrupted gating (leakage present); P74S did NOT cause leakage.

What was the final pathogenic classification from the study?

Both P74S and G155S are pathogenic, reclassifying VUS as clinically actionable.

What new biological insight came from these hypomorphic alleles?

They revealed close functional and spatial association between MKSR-2 and MKS-2 at the transition zone.

What is the mnemonic for the gating assay result comparing the two variants?

G155S = Gate broken (leakage present); P74S = Permission denied (no leakage, gate intac