In silico analysis of potential off-target sites to gene editing for Mucopolysaccharidosis type I using the CRISPR/Cas9 system: Implications for population-specific treatments.

Paola Carneiro,Martiela Vaz De Freitas,Ursula Matte,Shuang-Yong Xu

doi:10.1371/journal.pone.0262299

Paola Carneiro, Martiela Vaz De Freitas + Show 2 more

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https://doi.org/10.1371/journal.pone.0262299

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Abstract

Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase deficiency encoded by the IDUA gene. Therapy with CRISPR/Cas9 is being developed for treatment, however a detailed investigation of off-target effects must be performed. This study aims to evaluate possible off-targets for a sgRNA aiming to correct the most common variant found in MPS I patients (p.Trp402*). A total of 272 potential off-target sequences was obtained and 84 polymorphic sites were identified in these sequences with a frequency equal to or greater than 1% in at least one of the populations. In the majority of cases, polymorphic sites decrease the chance of off-target cleavage and a new PAM was created, which indicates the importance of such analysis. This study highlights the importance of screening off-targets in a population-specific context using Mucopolysaccharidosis type I as an example of a problem that concerns all therapeutic treatments. Our results can have broader applications for other targets already clinically in use, as they could affect CRISPR/Cas9 safety and efficiency.

Highlights

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by the deficiency of alpha-L-iduronidase (IDUA; EC 3.2.1.76), an enzyme coded by the IDUA gene
This work aims to evaluate possible off-target regions for a Streptococcus pyogenes guided by RNA (sgRNA) designed to correct the most common variant found in MPS I patients using the five most used in silico predictors for the CRISPR/Cas9 system [17]
After filtering for sequences with up to 6 mismatches and up to 2 indels, 272 sequences were obtained as potential off-target sites for this sgRNA, besides the on-target region

Summary

Introduction

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by the deficiency of alpha-L-iduronidase (IDUA; EC 3.2.1.76), an enzyme coded by the IDUA gene. Three syndromes are associated with the clinical spectrum from severe to attenuate: Hurler (OMIM #67014), Hurler-Scheie (OMIM #607015), Scheie (OMIM # 67016). Several pathogenic mutations were identified in the IDUA gene and p.Trp402 is the most common variant in MPS I patients in Western countries [2,3,4]. This null allele results in the absence of the gene product [5] and is present in all three disease forms. Therapy with Clustered Regularly Interspaced Short Palindromic Repeats associated with Cas proteins (CRISPR/Cas9) is under

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