CRISPR-based Gene Editing Protocol for Jurkat T Cells

Since 2007, the number of papers in the field of lncRNA biology has increased exponentially, demonstrating their role in a wide range of illnesses. However, problems like as low levels of expression and tissue-specific expression make it difficult to research their functional roles. One way to solve this is to play about with lncRNAs in vitro and see what happens to the cells. This methodology demonstrates how to overexpress lncRNAs by utilizing CRISPR technology to overexpress Interferon gamma antisense 1 (IFNG-AS1), which is linked to inflammatory bowel illness, in Jurkat T cells. In order to produce robust overexpression of various lncRNA splice variants at endogenous genomic loci, our strategy addresses transcription factor binding sites.

Experimental Materials and Recommended Services

Name	Comments	Related Products and Services
HEK293T cells	Transfection and viral particle production	293T Cell Line
Jurkat cells	Transduction and experimentation	Cell Line
cDNA synthesis kit	Synthesizes cDNA from RNA for gene expression analysis	miRNA cDNA Synthesis Kits MiqScript™ miRNA cDNA Synthesis Kit-10
Plasmid Purification Kit	Purifies bacterial DNA containing plasmids like dCas9 vector and gRNA plasmid	Construct Plasmid Purification Kits
RNA purification kit	Extracts RNA for cDNA synthesis and gene expression analysis.	RNA Purification Kit
dCas9 vector	Tool for genome editing and transcriptional regulation with guide RNAs	dCAS9-VP64_GFP pCDNA-dcas9-VP64 pdCas9-bacteria
gRNA plasmid	Contains guide RNA sequences for directing dCas9 to specific genomic loci	pgRNA-bacteria pgRNA-humanized pgRNA-modified
pMDG2.G	Plasmids for viral particle generation, encoding proteins for packaging and replication.	pMDLg/pRRE Plasmid DNA Production Service Linearized Plasmid GMP Production Plasmid Construction
pMDLg/pRRE	Plasmids for viral particle generation, encoding proteins for packaging and replication.

Procedure

Fig. 1 Illustrates a dual vector system for the overexpression of the lncRNA IFNG-AS1. (doi: 10.3791/59233) Figure 1. The IFNG-AS1 gene structure is schematically shown in (A), with the locations of guide RNA (gRNA) binding sites about the transcriptional start site (TSS) highlighted. The features of the gRNA and dCAS9 vectors are described in (B). (Rankin, C. R., et al.,2019)

1. Vector Design and Generation

a. Utilize online design tools (e.g., http://crispr-era.stanford.edu) to identify gRNA sequences. For this experiment, gRNA vectors were designed and created by a company, and the dCas9 plasmid was purchased online.

b. Verify that the gRNA sequence is situated within 100 base pairs of the transcriptional start point, upstream of the "NGG" nucleotide sequence. Make sure it is unique by utilizing genomic databases such as BLAT.

b. Keep the dCas9 and gRNA plasmids cold, at 4 ℃. Overnight at 37 °C, cultivate Escherichia coli on LB agar plates with ampicillin.

d. Spin down the LB bacterial culture and scale it up with ampicillin after shaking the bacteria all night.

e. Purify bacterial DNA using a plasmid purification kit.

f. For DNA purification, adhere to the resuspension, lysis, neutralization, equilibration, and elution procedures specified in the kit.

g. Complete the processes for column equilibration, DNA purification, washing, and elution.

h. Use isopropanol to precipitate DNA, followed by ethanol washing, air drying, and resuspension in water. Store DNA at -20 °C.

2. Virus Generation and Particle Count

a. Coat tissue culture dishes with poly-L-lysine and plate HEK 293T cells in complete DMEM overnight.

b. Transfect cells with plasmids containing virus components and dCas9 or gRNA vectors using calcium phosphate transfection.

c. Grow transfected cells and gather viral particle-containing supernatants.

d. To store viral particle aliquots, freeze them at -80 °C.

e. Using a p24 Enzyme-Linked Immunosorbent Assay (ELISA) kit, thaw and measure virus particles.

3. dCas9-VP64 Transduction

a. Culture Jurkat T cells in complete DMEM, spin down and resuspend in reduced serum media.

b. Plate cells and transduce with viral particles containing dCas9. Incubate at 37 °C with 5% CO2.

4. Selection and Clone Creation

a. Choose transduced cells, grow clonal cells, and place them in the puromycin-containing medium.

b. Plate cells for clone expansion and single-cell dilution.

b. To assess gene expression, extract RNA from clonal cells, create cDNA, and run real-time PCR.

5. gRNA Transduction, Selection, Clone Creation, and Screening

a. Retransduce Jurkat-dCas9 cells with gRNA-containing viruses and select cells with hygromycin.

b. Expand individual colonies, purify RNA, synthesize cDNA, and perform real-time PCR to analyze gene expression.

c. Determine fold change in gene expression using the comparative cycle threshold (Ct) method.

This protocol describes a textual and graphic strategy for lncRNA overexpression in vitro, showing that in a Jurkat T cell model, the lncRNA IFNG-AS1 linked to inflammatory bowel disease is overexpressed. This is accomplished by overexpressing a gene at an endogenous genomic locus using CRISPR technology. By targeting the gene's transcription start point, CRISPR technology may be used to achieve robust overexpression across a variety of lncRNA splice variants. The importance of long non-coding RNA (lncRNA) biology in a number of disorders is shown by the field's explosive growth in publications. Innovative methods like CRISPR-based overexpression offer important insights into the functional significance of lncRNAs in disease situations, despite obstacles like tissue selectivity and low expression levels.

* For research use only. Not intended for any clinical use.

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