Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Machine learning prediction of prime editing efficiency across diverse chromatin contexts.

The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates.

Enhancing prime editor activity by directed protein evolution in yeast.

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.