HAP1, a new revolutionary cell model for gene editing using CRISPR-Cas9

Abstract

The use of next-generation sequencing (NGS) technologies has been instrumental in the characterization of the mutational landscape of complex human diseases like cancer. But despite the enormous rise in the identification of disease candidate genetic variants, their functionality is yet to be fully elucidated in order to have a clear implication in patient care. Haploid human cell models have become the tool of choice for functional gene studies, since they only contain one copy of the genome and can therefore show the unmasked phenotype of genetic variants. Over the past few years, the human near-haploid cell line HAP1 has widely been consolidated as one of the favorite cell line models for functional genetic studies. Its rapid turnover coupled with the fact that only one allele needs to be modified in order to express the subsequent desired phenotype has made this human cell line a valuable tool for gene editing by CRISPR-Cas9 technologies. This review examines the recent uses of the HAP1 cell line model in functional genetic studies and high-throughput genetic screens using the CRISPR-Cas9 system. It covers its use in an attempt to develop new and relevant disease models to further elucidate gene function, and create new ways to understand the genetic basis of human diseases. We will cover the advantages and potential of the use of CRISPR-Cas9 technology on HAP1 to easily and efficiently study the functional interpretation of gene function and human single-nucleotide genetic variants of unknown significance identified through NGS technologies, and its implications for changes in clinical practice and patient care.

CRISPR/Cas9‐based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders

“…In fact, a recent review highlighted the usefulness of HAP1 as a valuable tool to study a wide range of diseases and physiological processes, including those related to oncology, virology, and metabolism, among others. 48 To assess the usefulness of the proposed method, we generated a total of 11 knock-in cell models, representing a diverse array of IMDs (Table 1). The feasibility of our methodological approach was first evaluated by testing the functional impact of seven pathogenic/likely pathogenic variants, in which disease-causality had been previously demonstrated at the functional level.…”

“…Moreover, an important percentage of genes involved in other Mendelian disorders are expressed in this cell line, indicating that the generation of KI HAP1 models could be appropriated for VUS evaluation not only in IMDs but also in a wide variety of genetic conditions provided that the associated phenotype is measurable in the cellular model. In fact, a recent review highlighted the usefulness of HAP1 as a valuable tool to study a wide range of diseases and physiological processes, including those related to oncology, virology, and metabolism, among others 48 …”

InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

“…eHAP cells, a fully haploid version of HAP1 cells, are useful in gene-function studies because its haploid genome requires editing only one allele to obtain a gene deletion (1, 2). Most gene-function studies also require the expression of transgenes.…”

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells

“…Although gene regulation of AGAT, which is physiologically expressed mainly in kidney, pancreas, and some brain cells, must be considered somewhat non-physiological in HAP1 cells, HAP1 cells are among the immortalized human cell lines expressing AGAT (as well as GAMT and CrT) and respond well to CT-mediated downregulation of AGAT expression. HAP1 cells represent a quasi-stable haploid cell line that has been used extensively in CRISPR-Cas9 based genome editing experiments to generate specific gene knockouts, targeting tagging of genes corresponding to the N- and C-termini of the protein coding region 33 , and in chemical and genetic screening campaigns 34 (and reviewed in 35 ). As such, we have used CRISPR homology- mediated repair 36 to tag AGAT with nanoluc luciferase (NLuc) in HAP1 cells to quantify and monitor the changes in AGAT-NLuc expression with increasing concentrations of extracellular CT.…”