Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity.

The current-generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A-to-G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off-target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library-assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off-target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4-7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off-target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease-suppressing mutations and disease-correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant-library-assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.

PMRD: a curated database for genes and mutants involved in plant male reproduction.

BACKGROUND: Male reproduction is an essential biological event in the plant life cycle separating the diploid sporophyte and haploid gametophyte generations, which involves expression of approximately 20,000 genes. The control of male reproduction is also of economic importance for plant breeding and hybrid seed production. With the advent of forward and reverse genetics and genomic technologies, a large number of male reproduction-related genes have been identified. Thus it is extremely challenging for individual researchers to systematically collect, and continually update, all the available information on genes and mutants related to plant male reproduction. The aim of this study is to manually curate such gene and mutant information and provide a web-accessible resource to facilitate the effective study of plant male reproduction. DESCRIPTION: Plant Male Reproduction Database (PMRD) is a comprehensive resource for browsing and retrieving knowledge on genes and mutants related to plant male reproduction. It is based upon literature and biological databases and includes 506 male sterile genes and 484 mutants with defects of male reproduction from a variety of plant species. Based on Gene Ontology (GO) annotations and literature, information relating to a further 3697 male reproduction related genes were systematically collected and included, and using in text curation, gene expression and phenotypic information were captured from the literature. PMRD provides a web interface which allows users to easily access the curated annotations and genomic information, including full names, symbols, locations, sequences, expression patterns, functions of genes, mutant phenotypes, male sterile categories, and corresponding publications. PMRD also provides mini tools to search and browse expression patterns of genes in microarray datasets, run BLAST searches, convert gene ID and generate gene networks. In addition, a Mediawiki engine and a forum have been integrated within the database, allowing users to share their knowledge, make comments and discuss topics. CONCLUSION: PMRD provides an integrated link between genetic studies and the rapidly growing genomic information. As such this database provides a global view of plant male reproduction and thus aids advances in this important area.

CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric ß0/ß0 transfusion-dependent ß-thalassemia.

Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate ß-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent ß-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the ß0/ß0 genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl-1 at screening to 15.0 and 14.0 g dl-1 at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.

Identification of allelic expression imbalance genes in human hepatocellular carcinoma through massively parallel DNA and RNA sequencing.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. The prognosis and treatment of this disease have changed little in recent decades because the mechanisms underlying most events of this disease remain obscure. Allelic variation of gene expression is associated with many important biological processes, which provide a new perspective to understand HCC pathogenesis at the molecular level. To identify allelic expression imbalance (AEI) genes in HCCs, we developed a computational method that considered accurate mapping and vigorous AEI detection using paired DNA-seq and RNA-seq data. We analyzed the DNA-seq and RNA-seq data derived from two HCC samples and two cell lines. By applying a strict criterion, a total of 203 tumor-specific AEI genes were identified with high confidence, and several genes have been reported to be associated with the migration or proliferation of cancer cells, such as the genes RELN and DHRS3. In addition, we also found some novel AEI genes in HCCs, such as HNRNPR and PTAFR. Our study provides new insight into AEI events that may contribute to understanding gene expression regulation, cell proliferation and migration, and tumorigenesis.

RNA over-editing of BLCAP contributes to hepatocarcinogenesis identified by whole-genome and transcriptome sequencing.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, although the treatment of this disease has changed little in recent decades because most of the genetic events that initiate this disease remain unknown. To better understand HCC pathogenesis at the molecular level and to uncover novel tumor-initiating events, we integrated RNA-seq and DNA-seq data derived from two pairs of HCC tissues. We found that BLCAP is novel editing gene in HCC and has over-editing expression in 40.1% HCCs compared to adjacent liver tissues. We then used RNA interference and gene transfection to assess the roles of BLCAP RNA editing in tumor proliferation. Our results showed that compared to the wild-type BLCAP gene, the RNA-edited BLCAP gene may stably promote cell proliferation (including cell growth, colony formation in vitro, and tumorigenicity in vivo) by enhancing the phosphorylation of AKT, mTOR, and MDM2 and inhibiting the phosphorylation of TP53. Our current results suggest that the RNA over-editing of BLCAP gene may serve as a novel potential driver in advanced HCC through activating AKT/mTOR signal pathway.