Over the last decade, next generation sequencing has become widely implemented in clinical practice. However, as genetic variants of uncertain significance (VUS) are frequently identified, the need for scaled functional interpretation of such variants has become increasingly apparent. One method to address this is saturation genome editing (SGE), which allows for scaled multiplexed functional assessment of single nucleotide variants. The current applications of SGE, however, rely on homology-directed repair (HDR) to introduce variants of interest, which is limited by low editing efficiencies and low product purity. Here, we have adapted CRISPR prime editing for SGE and demonstrated its utility in understanding the functional significance of variants in the NPC1 gene underlying the lysosomal storage disorder Niemann-Pick disease type C1 (NPC). Additionally, we have designed a genome editing strategy that allows for the haploidization of gene loci, which permits isolated variant interpretation in virtually any cell type. By combining saturation prime editing (SPE) with a clinically relevant assay, we have functionally scored and interpreted 256 variants in NPC1 haploidized HEK293T cells. To further demonstrate the applicability of this strategy, we used SPE and cell model haploidization to functionally score 465 variants in the BRCA2 gene. We anticipate that our work will be translatable to any gene with an appropriate cellular assay, allowing for more rapid and accurate diagnosis and improved genetic counselling and ultimately precise patient care.
Functional evaluation of novel molecules that promote stem cell mediated endogenous repair often require multiplexed in vivo transplant studies that are low throughput and hinder the rate of discovery. Here, we optimized and miniaturized a previously developed muscle endogenous repair (MEndR) in vitro assay that captures significant events of in vivo muscle endogenous repair to offer greater throughput for functional validation studies. The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), prospectively isolated from mouse skeletal muscle tissue, are engrafted onto the engineered template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. We evaluated two different commercially available human primary myoblast lines and were able to successfully generate miniaturized 3D muscle templates, as well as recapitulate the in vivo outcomes of a known modulator of MuSC mediated repair but only in the presence of both the stem cells and the regenerative milieu. Thus, the mini-MEndR culture assay captures the ability of different molecular treatments to modulate donor MuSC skeletal muscle production and niche repopulation. The miniaturized predictive assay offers a simple, scaled platform with which to investigate MuSC endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.
Over the last decade, next generation sequencing has become widely implemented in clinical practice. However, as genetic variants of uncertain significance (VUS) are frequently identified, the need for scaled functional interpretation of such variants has become increasingly apparent. One method to address this is saturation genome editing (SGE), which allows for scaled multiplexed functional assessment of single nucleotide variants. The current applications of SGE, however, rely on homology-directed repair (HDR) to introduce variants of interest, which is limited by low editing efficiencies and low product purity. Here, we have adapted CRISPR prime editing for SGE and demonstrated its utility in understanding the functional significance of variants in the NPC1 gene underlying the lysosomal storage disorder Niemann-Pick disease type C1 (NPC). Additionally, we have designed a genome editing strategy that allows for the haploidization of gene loci, which permits isolated variant interpretation in virtually any cell type. By combining saturation prime editing (SPE) with a clinically relevant assay, we have functionally scored and interpreted 256 variants in NPC1 haploidized HEK293T cells. To further demonstrate the applicability of this strategy, we used SPE and cell model haploidization to functionally score 465 variants in the BRCA2 gene. We anticipate that our work will be translatable to any gene with an appropriate cellular assay, allowing for more rapid and accurate diagnosis and improved genetic counselling and ultimately precise patient care.
Development of a repeatable method for delivering transgene payloads to human induced pluripotent stem cells (hiPSCs) without risking unintended off-target effects is not fully realized. Yet, such methods are indispensable to fully unlocking the potential for applying synthetic biological approaches to regenerative medicine, delivering quantum impacts to cell-based therapeutics development. Here we present a toolkit for engineering hiPSCs centred on the development of two core ‘landing-pad’ cell-lines, facilitating rapid high-efficiency delivery of transgenes to theAAVS1safe-harbour locus using the Bxb1 large-serine recombinase. We developed two landing-pad cell lines expressing green and red fluorescent reporters respectively, both retaining stemness whilst fully capable of differentiation into all three germ layers. A fully selected hiPSC population can be isolated within 1-2 weeks after landing-pad recombinase-mediated cassette exchange. We demonstrate the capability for investigator-controlled homozygous or heterozygous transgene configurations in these cells. As such, the toolkit of vectors and protocols associated with this landing-pad hiPSC system has the potential to accelerate engineering workflows for researchers in a variety of disciplines.
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