Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery.

Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined in vitro differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved definitive endoderm and hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End-stage hepatocytes integrated into three-dimensional (3D) liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End-stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without FA supplementation, recapitulating a feature of NASH hepatocytes in vivo Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.

Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.

Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.

High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.