Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant

Figures & data

Figure 1. Overview of the three retinal isoforms of CRB1 and its location. All isoforms share exon 6. CRB1-B is the most abundant retinal isoform. It has a unique N-and C-terminus while sharing the transmembrane domain as well as a large part of the extracellular domain with CRB1-A. the patient’s novel nonsense mutation affects CRB1-A and CRB1-B, while the missense mutation affects all retinal CRB1 isoforms. CRB1 is thought to play an important role in maintaining the integrity of the OLM.

Figure 2. Clinical phenotype of patient with compound heterozygous for single nucleotide variants in CRB1. (a) Infrared (left) and autofluorescence (right) imaging shows atrophic changes in the central retina with hyperfluorescent spots outside the temporal arcades. (b) Characteristic for a CRB1-associated RP, both left and right eye show increased retinal thickness. the retinal lamination pattern is coarse, reminiscent of an immature retina and disrupted by intraretinal exudates.

Table 1. Classification of the novel CRB1 c.2885T>A (p.Leu962Ter) and CRB1 c.2056C>T (p.Arg686Cys) variant using ACMG standards and guidelines.

CRB1 c.2885T>A (p.Leu962Ter)
Evidence of pathogenicity	Category
Very Strong	PVS1: nonsense variant predicted to lead to NMD and LOF in CRB1 in two of the most abundant and important CRB1 isoforms (CRB1-A and CRB1-B)
Moderate	PM2: the variant is absent from controls in gnomAD, ExAC and 1000 G databases
Supporting	PP4: patient’s phenotype of early-onset RP with thickened retina is highly specific for a CRB1-associated IRD
→pathogenic variant
CRB1 c.2056C>T (p.Arg686Cys)
Evidence of pathogenicity	Category
Moderate	PM3: this missense variant has been reported to occurs with pathogenic variants in this case report as well as in Wang et al. (50) and Liu et al. (51). In both instances, phase is unknown.
	PM2: a single heterozygous occurrence in >250,000 alleles in gnomAD.
Supporting	PP3: three lines of computational evidence (Polyphen2 and Mutation Taster) predict a deleterious effect of this missense variant located in the highly preserved Arginine at amino acid position 686 for all protein-coding CRB1 isoforms.
	PP4: patient’s phenotype of early-onset RP with thickened retina is highly specific for a CRB1-associated IRD.
→likely pathogenic variant

NMD: nonsense mediated decay; LOF: loss of function; gnomAD: Genome Aggregation Database; ExAC: Exome Aggregation Consortium; 1000 G: 1000 Genes; PolyPhen2: Polymorphism Phenotyping version 2; SIFT: Sorting Intolerant from Tolerant; RP: retinitis pigmentosa; IRD: inherited retinal disease.

Figure 3. Therapeutic “read-through” approach mediated by CRISPR-pass. While base editing with CRISPR-pass does not allow a true ribosomal read-through, nonsense mutations not amenable to conventional base editing strategies can be turned into a missense change by using an ABE-xCas base editing construct (CRISPR-pass), targeting the coding or non-coding strand. in this case, a bystander mutation would occur when targeting the coding strand due to the presence of an additional a in the editing window. the tryptophan and glutamine missense changes resulting from CRISPR-pass mediated editing are predicted to be “possibly damaging” by the in-silico tool Polyphen 2, which evaluates the functional impact of missense changes.

Figure 4. Overview of base editing strategy for a C>T transition mutation. the patient could be transferred to a carrier status by using an ABE7.10 or an ABE8 fused to a SaCas9-KKH, which displays a relaxed PAM site 5’-NNNRRT-3’ and an editing window within 2–15 nucleotides upstream of the PAM site. No additional A’s are present in the editing window, greatly reducing the possibility of bystander mutations.

Figure 5. Decision making tree for developing a base editing strategy for a patient with compound heterozygous mutation in CRB1.

Wang L, Zhang J, Chen N, Wang L, Zhang F, Ma Z, Li G, Yang L, et al. Application of whole exome and targeted panel sequencing in the clinical molecular diagnosis of 319 Chinese families with inherited retinal dystrophy and comparison study. Genes (Basel). 2018;9(7):360. doi:10.3390/genes9070360.

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Liu X, Tao T, Zhao L, Li G, Yang L.Molecular diagnosis based on comprehensive genetic testing in 800 Chinese families with non-syndromic inherited retinal dystrophies. Clin Exp Ophthalmol. 2021;49(1):46–59. doi:10.1111/ceo.13875.

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