Functional rescue of F508del-CFTR through revertant mutations introduced by CRISPR base editing.

Cystic Fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in the CFTR gene, resulting in functional impairment of the encoded ion channel. F508del mutation, a trinucleotide deletion, is the most frequent cause of CF affecting approximately 80% of persons with cystic fibrosis (pwCFs). Even though current pharmacological treatments alleviate the F508del-CF disease symptoms there is no definitive cure.

The NIPBL-gene mutation of a Cornelia de Lange Syndrome patient causes deficits in the hepatocyte differentiation of induced Pluripotent Stem Cells via altered chromatin-accessibility.

The Cornelia de Lange syndrome (CdLS) is a rare genetic disease, which is characterized by a cohesinopathy. Mutations of the NIPBL gene are observed in 65% of CdLS patients. A novel iPSC (induced Pluripotent Stem Cell) line was reprogrammed from the leukocytes of a CdLS patient carrying a missense mutation of the NIPBL gene.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines.

CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species.

Eukaryotic-driven directed evolution of Cas9 nucleases.

Background: Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells.