CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors

Abstract

A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

“…First, these forty constructs were evaluated for their on-target DNA editing efficiencies relative to ABE7.10 across eight genomic sites containing target A bases in positions ranging from 2 to 20 (where NGG PAM = positions 21,22,23) within the canonical 20-nt S. pyogenes protospacer ( Supplementary Fig. 5).…”

“…Another class of undesired ABE-mediated genome edits at an on-target locus can be an ABE-dependent cytosine to uracil (C•G to T•A) conversions 16,22 . At the eight target sites tested, we measured the 95 th percentile of C-to-T editing to be 0.45% with ABE8 variants and 0.15% with ABE7.10-d or -m, indicating that on-target cytosine deamination with ABEs can occur but the frequencies are generally very low ( Supplementary Fig.…”

CRISPR-Cas9 DNA Base-Editing and Prime-Editing

“…Targeted base-editing restored dystrophin expression in 17% of myofibers, a level which is sufficient to improve muscle function. Lee et al developed CRISPR-pass, a targeted tool for bypassing premature termination codons using CRISPR-mediated adenine base-editors that showed systematic rescue of all possible cases of PTCs [ 12 ]. This approach is applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database.…”

“…Moreover, base-editing can be applied to autosomal dominant diseases, where gene augmentation is not a suitable approach due to the requirement for silencing or ablating the defective gene in autosomal dominant diseases. In addition, recently developed base-editor adaptations such as “CRISPR-Pass” may prove advantageous in the correction of diseases characterized by premature terminations or alternative splicing [ 12 ]. Indeed, the therapeutic potential of DNA base-editors and prime-editors is tremendous [ 8 , 9 , 13 ].…”

Dead Cas Systems: Types, Principles, and Applications

“…Editing codons with nonsense mutations using CRISPR-Pass helps to recover production of active proteins. CRISPR-Pass was predicted to correct up to 95% mutations described in ClinVar database [160]. dCas-base editors were patented as antiviral tools capable of introducing mutations into viral genomes to block replication and protein synthesis of viruses including HIV, HBV, human papilloma virus, and Epstein–Barr virus.…”