Cas9 Variant Screening
Cas9 Variant Screening - Related Videos
12:25
Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells
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2026
Here, we present a protocol for generating chemoresistant cells using a dose-escalation approach, followed by a CRISPR/Cas9-based screen using a focused sgRNA library targeting epigenetic modifiers to identify regulators of acquired chemoresistance. The protocol also provides multiple optimization points tailored to chemoresistant cell models, offering a robust framework for researchers investigating resistance mechanisms.
02:30
Histone Variants at the Centromere
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2020
Histone variants are the histone proteins with structural and sequence variations. These variants may be regarded as “mutant” forms that replace their canonical histone counterparts in the nucleosomes. Specific post-translational modifications on the histone variants enable further chromatin complexity and regulate tissue-specific gene expression. The most common histone variants are from histone H2A, H2B, and linker histone H1 families. However, several variants of histone H3 variants are also...
11:35
Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
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Cited by 15 •
2016
We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.
02:30
A CRISPR-Cas9 Technique for Gene Editing in T Cells
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2025
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell's non-homologous end-joining mechanism, leading to gene knockout.
02:55
Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System
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2025
The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.
FAQs
PAM compatibility determines whether a variant can recognize a target site adjacent to the required protospacer-adjacent motif. Variants with different PAM preferences can therefore differ in which genomic loci are addressable, even when the guide RNA and target region are otherwise suitable. Screening this property is especially relevant when a desired locus lacks compatibility with the original enzyme.
Matched guide RNAs and target sequences make activity comparisons interpretable. If those inputs change between variants, differences in cleavage could reflect the guide or DNA substrate rather than the enzyme. Keeping the pairing consistent allows investigators to compare cleavage activity and editing efficiency directly, while separate target sequences can test whether performance depends on locus context.
A useful screen considers precision alongside activity. Reporter assays or sequencing can indicate whether a variant modifies the intended target, whereas targeted or genome-wide analyses can reveal unintended sites. Comparing these outcomes distinguishes strong on-target editing from activity that reaches additional sequences, helping genetics researchers prioritize variants with a more controlled editing profile.
An evaluation typically pairs each Cas9 variant with a guide RNA and selected target sequences, measures cleavage or editing, and compares results across variants. The readout may then be examined for PAM compatibility, efficiency, and unintended effects. This sequence of comparisons converts individual measurements into a basis for choosing an enzyme for a specific locus.
Reporter systems offer a screening format for comparing variant activity, while targeted sequencing examines editing at selected DNA sites. Genome-wide analyses extend the assessment to unintended modifications across the genome. The appropriate readout depends on whether the immediate goal is broad comparison, confirmation at a defined locus, or a more comprehensive evaluation of editing specificity.
Selection depends on the requirements of the intended experiment. A variant may be favored because it reaches a locus with otherwise unsuitable PAM compatibility, produces stronger editing, or shows fewer unintended effects. These comparisons support more reliable functional studies involving gene disruption or repair and can also inform evaluation of candidates for potential therapeutic development.