Abstract
Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.
Highlights
The field of genome engineering was revolutionised by the harnessing of the bacterial immune system CRISPR/CRIPSR associated (Cas) to create programmable DNA double strand breaks in the human genome with an unprecedented ease and adaptability [1]
Because U is read as a thymine (T) by the DNA polymerase during mismatch repair, cytosine base editors (CBEs) are able to catalyse a C:G to T:A transitions, within a 5 bp editing window in the single stranded DNA (ssDNA) bubble created by Streptococcus pyogenes’ Cas9 (SpCas9) [2]
Developing an adenine base editor (ABE) that would enable A:T to G:C edits was attractive, since C>T single nucleotide variants (SNVs) account for over half of human pathogenic SNV recorded in the ClinVar database and are the most common human pathogenic SNV [3]
Summary
The field of genome engineering was revolutionised by the harnessing of the bacterial immune system CRISPR/Cas to create programmable DNA double strand breaks in the human genome with an unprecedented ease and adaptability [1]. GBE are the first to enable editing of C:G to G:C transversion mutations in mammalian cell lines [9,10,11] This is achieved by making use of the endogenous cellular base excision repair (BER) pathway and fusing a uracil DNA glycosylase (UDG) enzyme to an APOBEC deaminase. This catalyses excision of the U and creation of an abasic site followed by mutagenesis across this abasic site [9,10,11]. While we have included GBE in this analysis, DNA off-target effects as well as the reason behind different editing activities across target sites are still outstanding for these constructs
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