Biology subjects

Majeti, K. R.

Publications and source records attributed to Majeti, K. R..

3 recordsLinked to original sources

bioRxiv2024

Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media--EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

synthetic biology↗

bioRxiv2023

Using human genetics to develop strategies to increase erythropoietic output from genome-edited hematopoietic stem and progenitor cells

Human genetic polymorphisms result in a diversity of phenotypes. Some sequences are pathologic and lead to monogenic diseases, while others may confer beneficial traits. Genome editing is a powerful tool to recreate genotypes found in the population, including the ability to correct pathologic mutations. One of the best characterized naturally occurring mutations causing congenital erythrocytosis arises from a truncation in the erythropoietin receptor (tEPOR) which can result in non-pathogenic hyper-production of red blood cells (RBCs). Using the precision of CRISPR/Cas9 genome editing, we have recreated tEPOR and studied the effect of variations of the genotype on RBC development. We then combined tEPOR with a correction strategy developed for {beta}-thalassemia and demonstrated that coupling the two genome editing events gave RBCs a significant selective advantage. This demonstrates the potential of combining human genetics with the precision of genome editing to enable safer and more effective genome editing therapies for patients with serious genetic diseases.

genetics↗

bioRxiv2022

Comparative analysis of CRISPR off-target activity discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells

While CRISPR-based editing most often occurs at DNA sequences with perfect homology to the guide RNA (gRNA), unintended editing can occur at highly homologous regions (i.e., off-target (OT) sites). Due to the pace at which genome editing therapies are approaching clinical applications, there is an emerging need to define effective workflows for investigating OT editing effects. A number of homology-dependent, in silico-based prediction methods and wet lab-based empirical methods exist to investigate OT editing, but few have been subjected to analytical assessment or head-to-head comparison in human primary cells using an ex vivo editing process optimized for high-fidelity gene editing. Therefore, we sought to compare publicly available in silico tools (COSMID, CCTop, and Cas-OFFinder) as well as empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) in the context of ex vivo hematopoietic stem and progenitor cell (HSPC) editing. To do so, we edited CD34+ HSPCs using 11 different guide RNAs (gRNAs) complexed with HiFi Cas9, then performed targeted next-generation sequencing of ~200-site panels containing a range of nominated OT sites identified by in silico and empirical methods. We identified an average of 0.45 OT sites per gRNA at an indel detection limit of 0.5%. This study confirmed the marked improvement in specificity with HiFi Cas9 compared to wild-type Cas9 without compromising on-target activity when delivered as an RNP. Additionally, all HiFi Cas9 OT sites using a standard 20nt gRNA were identified by all OT detection methods with one exception (SITE-seq did not identify an OT generated by an AAVS1 gRNA). This resulted in high sensitivity for the majority of OT nomination tools, however due to the large number of false positives called by most methods, in silico-based COSMID and empirical methods DISCOVER-Seq and GUIDE-Seq attained the highest positive predictive value. We did not find the empirical methods identified off-target sites that were not also identified by bioinformatic methods when delivered as an RNP complex. Finally, this study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity as well as positive predictive value which would enable more efficient identification of potential off-target sites without compromising a thorough examination for any given gRNA.

genomics↗