Build CRISPR/Cas9 Plasmid in 5 steps
What does CRISPR stand for?
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Prime editing is an evolved CRISPR, an earlier technology for genome editing. CRISPR searches the defect and then removes it from DNA in a sort of finding and deleting function in software for the word processing. At least, this is what the theory suggests but this has shown a lot of risks than being a help.
ThermoFisher Scientific explains a way to build your plasmid in the 5 Steps:
Step 1: Design
The CRISPR-Cas9 system consists of the CRISPR-gRNA and the Cas9 nuclease, which are target-specific. The Cas9 and gRNA must be expressed together in the target cells in order to be effective in genome editing. They offer the necessary instruments to design and create plasmid expression plasmids specific to the GRNA and Cas9 targets that allow you to pursue various experimental strategies.
Products for CRISPR-Cas9 design
For designing your vector you may choose between two separate plasmids for Cas9 and gRNA, respectively, or a single plasmid for both genes:
- Plasmid for Cas nuclease expression obtained from open-access resources (e.g. Addgene, the nonprofit global plasmid repository)
- gRNA cloning vector obtained from open-access resources (e.g. Addgene, the nonprofit global plasmid repository)
- GeneArt CRISPR Nuclease vector for cloning Cas9 and gRNA together
For constructing your own plasmid for gRNA expression you may need:
- Oligonucleotides containing gRNA sequence
- Anza Restriction and Cloning Enzymes including Type II S restriction enzymes and ligase
For easy cloning of novel CRISPR nuclease sequences or subcloning work you may need:
- Invitrogen Platinum SuperFi II DNA Polymerase (additional product formats are available)
- Zero Blunt TOPO PCR Cloning Kit (additional products for TOPO blunt-end subcloning are available)
Step 2: Transform
The vector can be transmitted to E. coli after cloning DNA fragments for device components of CRISPR-Cas9. The cells of E. coli to achieve plasmid expression in adequate quantities. They sell a wide range of competent E. coli. The selection of E. coli cells depends on the method of transformation and on your experiment ‘s efficiency.
There are numerous methods to test if the plasmid structure is right, i.e. PCR, digestion or sequence restriction. The decision depends on whether you want to know if the plasmid contains the DNA insert, whether the insert is in the right direction or if the insert is correctly sequenced. We provide tools for molecular biology to apply any form of research you choose.
Products used for plasmid transformation and subsequent screening of clones
For the efficient transformation of constructed CRISPR plasmids, you need competent E. coli cells. We recommend using One Shot MAX Efficiency DH5a T1R Chemically Competent Cells (additional products and sizes are available).
Isolate plasmid DNA in sufficient quantities and maximal purity for subsequent delivery to eukaryotic cells. Use PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit to generate high yields of the endotoxin-free plasmid (additional products and sizes are available).
Before proceeding into following steps you may wish to verify that your plasmid constructs are correct. Chose between different analysis methods:
- Invitrogen Platinum II Hot-Start Green PCR Master Mix (2X) for fast colony screening by PCR (additional product formats are available)
- Anza Restriction Enzymes for restriction analysis of plasmid DNA
- Sanger sequencing reagents for sequence analysis
Step 3: Deliver
CRISPR-Cas9 allows genome editing very simple and is very effective in wide applications such as stem-cell technique, gene therapy, models for tissues and animal diseases and developing transgenic plants that are disease-resistant.
Transfection is the process of introducing DNA, mRNA or protein systems into eukaryotic cells through CRISPR-Cas9. Constitutes vary extensively in delivery technologies, including the transfection of lipid nanoparticles, viral supply, and physical techniques such as electroporation. Build CRISPR/Cas9 Plasmid in 5 steps
Products used for CRISPR construct delivery into eukaryotic cells
- Lipofectamine transfection reagents are used for easy DNA and RNA delivery to a broad range of cell types by lipid nanoparticle-mediated transfection
- Neon Transfection System is used for constructing introduction into difficult-to-transfect eukaryotic cells by means of electroporation
- Lipofectamine CRISPRMAX Cas9 reagent – a premium solution for delivering our TrueCut Cas9 v2 and TrueGuide Synthetic sgRNA
Cell Culture Tools and Essentials:
- Sera – for your specific cell culture needs—from basic research to speciality assays
- Cell Culture Plastics – for optimum cell growth and consistency in cell culture
- Cell Culture Essentials – find the tools and resources you need for successful cell culture
Step 4: Detect
Any genome-editing technique you use will help you achieve robust and consistent results with careful monitoring. Begin with specific cell counts and viability tests, then test and confirm the cell genotype. The mutant sequence genotyping approach is based on the mutant form introduced by the CRISPR-generated edit. The most popular strategies are as follows:
- Improved PCR and gel electrophoresis for broader indels detection
Mismatch-cleavage assay for Indel detection (T7 Endonuclease I cleavage assay)
Digestion and amplification of PCR for the study of HDR
PCR amplification and cloning followed by Sanger sequencing
Enhanced PCR and NGS
Products used to detect CRISPR-mediated genome modifications
- Invitrogen Platinum Universal Direct PCR Master Mix for amplification of target DNA directly from crude samples of CRISPR-edited cells, without requiring DNA purification. Subsequent amplicons can be subjected to restriction digestion and analysis on the gel or cloned and sequenced
- Invitrogen Platinum II Hot-Start Green PCR Master Mix (2X) for fast PCR amplification of the target genomic area that later could undergo restriction digestion and subsequent analysis on the gel (additional product formats are available)
- Invitrogen Platinum SuperFi II PCR Master Mix for accurate amplification of the target genomic area followed by mismatch analysis within DNA fragments, or amplicon cloning and sequence analysis of the clones (additional product formats are available)
- Anza Restriction Enzymes for restriction analysis of PCR fragments to detect genome modifications
- Genomic cleavage detection kit for the detection of mutated sequences by mismatch-cleavage analysis of PCR-amplified genomic target regions
- Zero Blunt TOPO PCR Cloning Kit for Sequencing to create clones carrying DNA fragments with sequences from genomic targets for subsequent Sanger sequencing (additional products and sizes are available)
- GeneJET Plasmid Miniprep Kit to isolate plasmid DNA from E. coli clones, later analyzed by sequencing (additional products and sizes are available)
- E-Gel Power Snap Electrophoresis System Starter Kit, EX 2% for rapid, real-time DNA analysis on a gel (additional products and sizes are available)
- Sanger sequencing for sequence analysis of the clones
- DNA oligonucleotide primers for PCR
- PCR Tubes, Plates & Accessories
Step 5: Characterize
For knockout, knock-in, or gene expression modulation, CRISPR is routinely used and the effects can be measured using cell analysis techniques. Real-time PCR allows gene-size monitoring of changes in expression, for example, as the non-meaning mediated decay reduces transcript levels, whereas Western blotting is used to detect protein expression changes in the cell population. Imaging facilitates the direct study of protein expression, cell morphology and compartmentalisation changes, while high content analysis (HCA) provides quantitative rigour in the automation of the imaging process.
Products used for further CRISPR analysis and edited cell collection
- For the expression analysis by real-time PCR isolate transcript RNA from the edited cells and transcribe them into cDNA:
- For the differential gene expression detection, you may perform whole transcriptome sequencing. We recommend preparing cDNA libraries by using Collibri Library Prep kits for RNA-seq kit that enables strand-specific RNA sequencing on Illumina next-generation sequencing (NGS) systems
- Attune NxT Flow Cytometer for multiparameter analysis of individual cells
- For the confidence in every analysis, method use Ultra Pure Dnase/Rnase-Free Distilled Water