Validation of a CRISPR-engineered NHIP knockout neural cellular model via Nanopore Sequencing
Presentation Type
Oral Presentation
Presentation Type
Submission
Keywords
NHIP, nanopore, knockout, brain, ASD, CRISPR, cell, clone, genome, mutation
Department
Biology
Major
Biology
Abstract
Autism spectrum disorder (ASD) is influenced by genetic and environmental factors that affect neuronal development. We previously discovered a gene differentially expressed in the placenta of children who later develop autism, which we named Neuronal Hypoxia Inducible, Placental Associated (NHIP) which opens a new avenue for research and intervention.
The objective of this study was to validate an NHIP knockout neural cell model. Specifically, our goal was to assess the specificity of CRISPR/Cas9 to mutate only the NHIP locus, without introducing unintended mutations. The method we employed was nanopore sequencing. Briefly, genomic DNA was first isolated from LUHMES neural cell clones, including a wild-type control, a predicted heterozygous knockout clone, and a predicted homozygous NHIP knockout clone. DNA quality was then assessed using Qubit fluorometric quantification to determine purity and concentration. Next, high-molecular-weight genomic DNA was fragmented using Covaris g-TUBEs, and fragment size was verified by agarose gel electrophoresis. Finally, DNA nicks were repaired and end-ligated before libraries were loaded on a nanopore flow cell. Long-read sequencing enabled us to analyze the NHIP locus and genomic regions that are highly complementary to the guide RNA (gRNA) used for NHIP targeting. The results of our sequencing of the NHIP gene confirmed the absence of regions of interest. The data demonstrate that nanopore sequencing effectively validates NHIP knockout cellular models and confirms targeted genome edits while assessing potential off-target mutations. Future research will be done on observing how using this validated cell model, we will be able to directly assess the role of NHIP in neuronal cell differentiation.
Faculty Mentor
Antonio Gomez
Funding Source or Research Program
Academic Year Undergraduate Research Initiative
Location
Black Family Plaza Classroom 189
Start Date
10-4-2026 2:45 PM
End Date
10-4-2026 3:00 PM
Validation of a CRISPR-engineered NHIP knockout neural cellular model via Nanopore Sequencing
Black Family Plaza Classroom 189
Autism spectrum disorder (ASD) is influenced by genetic and environmental factors that affect neuronal development. We previously discovered a gene differentially expressed in the placenta of children who later develop autism, which we named Neuronal Hypoxia Inducible, Placental Associated (NHIP) which opens a new avenue for research and intervention.
The objective of this study was to validate an NHIP knockout neural cell model. Specifically, our goal was to assess the specificity of CRISPR/Cas9 to mutate only the NHIP locus, without introducing unintended mutations. The method we employed was nanopore sequencing. Briefly, genomic DNA was first isolated from LUHMES neural cell clones, including a wild-type control, a predicted heterozygous knockout clone, and a predicted homozygous NHIP knockout clone. DNA quality was then assessed using Qubit fluorometric quantification to determine purity and concentration. Next, high-molecular-weight genomic DNA was fragmented using Covaris g-TUBEs, and fragment size was verified by agarose gel electrophoresis. Finally, DNA nicks were repaired and end-ligated before libraries were loaded on a nanopore flow cell. Long-read sequencing enabled us to analyze the NHIP locus and genomic regions that are highly complementary to the guide RNA (gRNA) used for NHIP targeting. The results of our sequencing of the NHIP gene confirmed the absence of regions of interest. The data demonstrate that nanopore sequencing effectively validates NHIP knockout cellular models and confirms targeted genome edits while assessing potential off-target mutations. Future research will be done on observing how using this validated cell model, we will be able to directly assess the role of NHIP in neuronal cell differentiation.