Mutant allele knockout with novel CRISPR nuclease promotes myelopoiesis in ELANE neutropenia.

Severe congenital neutropenia (SCN) is a life-threatening marrow failure disorder, usually caused by heterozygous mutations in ELANE. Potential genetic treatment strategies include biallelic knockout or gene correction via homology-directed repair (HDR). Such strategies, however, involve the potential loss of the essential function of the normal allele product or limited coverage of diverse monogenic mutations within the patient population, respectively. As an alternative, we have developed a novel CRISPR-based monoallelic knockout strategy that precisely targets the heterozygous sites of single-nucleotide polymorphisms (SNPs) associated with most ELANE mutated alleles. In vitro studies demonstrate that patients' unedited hematopoietic CD34+ cells have significant abnormalities in differentiation and maturation, consistent with the hematopoietic defect in SCN patients. Selective knockout of the mutant ELANE allele alleviated these cellular abnormalities and resulted in about 50%-70% increase in normally functioning neutrophils (p < 0.0001). Genomic analysis confirmed that ELANE knockout was specific to the mutant allele and involved no off-targets. These results demonstrate the therapeutic potential of selective allele editing that may be applicable to SCN and other autosomal dominant disorders.

Structure and function of photosystem I in Cyanidioschyzon merolae.

The evolution of photosynthesis from primitive photosynthetic bacteria to higher plants has been driven by the need to adapt to a wide range of environmental conditions. The red alga Cyanidioschyzon merolae is a primitive organism, which is capable of performing photosynthesis in extreme acidic and hot environments. The study of its photosynthetic machinery may provide new insight on the evolutionary path of photosynthesis and on light harvesting and its regulation in eukaryotes. With that aim, the structural and functional properties of the PSI complex were investigated by biochemical characterization, mass spectrometry, and X-ray crystallography. PSI was purified from cells grown at 25 and 42 °C, crystallized and its crystal structure was solved at 4 Å resolution. The structure of C. merolae reveals a core complex with a crescent-shaped structure, formed by antenna proteins. In addition, the structural model shows the position of PsaO and PsaM. PsaG and PsaH are present in plant complex and are missing from the C. merolae model as expected. This paper sheds new light onto the evolution of photosynthesis, which gives a strong indication for the chimerical properties of red algae PSI. The subunit composition of the PSI core from C. merolae and its associated light-harvesting antennae suggests that it is an evolutionary and functional intermediate between cyanobacteria and plants.