Correction of a Factor VIII genomic inversion with designer-recombinases

Abstract

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

The bacteriophage lambda integrase catalytic domain can be modified to act with the regulatory domain as a recombination-competent binary recombinase

“…Since the main difference between the two vector designs is the relative location of the strong constitutive promoter, it is reasonable to assume that it is the ‘upstream’ location of this promoter that makes the positive reporter configuration less recombinationally active. The promoter location differently affects the topology of DNA upstream and downstream of a transcription bubble: DNA is overwound downstream of the bubble while it is underwound upstream of it ( 24 ). Since in the experiments with the negative reporter (in which the 5′ recombination target likely experiences underwinding) Int was able to generate the correct recombination outcome with all recombination pairs tested, we hypothesize that it is the recombination target underwinding that positively affects the recombination efficiency.…”

Ameliorative Impacts of Dactyloctenium aegyptium and Parapholis incurva Ethanolic Extracts on Sodium Fluoride Induced Pathological Alterations in the Thyroid Gland in Male Rats

“…Calcium serum levels were raised as a result of thyroid hormone problems. Enhanced blood calcium concentration stimulates calcitonin secretion [4], which appears to be the cause of increased calcitonin cytoplasmic reactivity in parafollicular cells.…”

“…Small C cells, also known as parafollicular cells, are found between thyroid follicles and secrete polypeptide hormone known as calcitonin. Calcitonin secretion is triggered by a rise in serum calcium levels, and calcitonin protects against hypercalcemia [4]. Calcium is necessary for various intracellular and extracellular activities as well as skeletal support in the body [5].…”

Quantification of evolved DNA-editing enzymes at scale with DEQSeq

“…By simultaneously capturing the target site sequence with the enzyme sequence on the same DNA fragment, the DNA editing rate for each enzyme variant can be quantified. We demonstrate the applicability of this platform on two different DNA editing systems, namely designer-recombinases [ 5 , 10 – 15 ] and evolved Cas12f-derived [ 16 – 19 ] mini-ABEs.…”