CRISPR Genome Editing
Clustered regularly interspaced short palindromic repeats, more colloquially known as CRISPR, are a genetic component of bacterial and archaeal antiviral immunity. They are composed of a series of palindromic repeats and spacer sequences. Spacer sequences are derived from the genomes of bacteriophages that have invaded the cell before making them a type of adaptive immunity. Many proteins related to this immune system are commonly referred to as CRISPR-associated (Cas) proteins and they allow the cell to generate and use spacer sequences.
Different Cas endonucleases and guide RNA (gRNA) form the core of CRISPR-Cas genome editing. A gRNA is composed of a scaffold sequence, which allows it to bind to a specific Cas protein, and an artificial spacer homologous to the sequence of interest. One of the most commonly used Cas proteins is Cas9 endonuclease: when bound to any gRNA strand it locates a homologous sequence and cuts both of the strands at a relatively specific location.
The CRISPR-Cas9 machinery can be delivered into cells in several ways, depending on the project needs. These include:
Nucleofection of in vitro -assembled Cas9-guideRNA ribonucleoprotein (RNP) complexes (non-viral gene editing)
Transient transfection of Cas9 and guide RNA -encoding plasmids
Viral transduction of Cas9 and guide RNA -encoding plasmids
The Genome Editing Core provides services for non-viral gene editing, and also offers consultation for other delivery methods.
CRISPR Gene Knock Out
In CRISPR-Cas mediated knockout, an active Cas endonuclease (e.g. Cas9) and a gRNA designed to target the sequence of interest are introduced to a cell. Cas9 will eventually cut at the homology sequence and the cell will attempt to repair the damage done. The repair mechanism eukaryotic cells employ are not always accurate, and some small indels or SNPs can form at the cutting site. If these changes cause a frameshift or a nonsense stop codon, the gene product cannot be expressed.
Gene-specific gRNA design
Preparation of the Cas9 RNP and nucleofection to cells of interest
Analysis of protein knock-out efficiency by western blotting or flow cytometry
Analysis of guide efficiency in the DNA level (TIDE analysis)
Please, do not hesitate to contact Genome Editing Core for consultation or general study design.
When provided with a repair template, gRNA-targeted double-strand breaks created by the Cas9 enzyme can be repaired through Homology-directed repair (HDR) mechanism, allowing for introduction of genetic sequences or mutations into specific genomic loci. This can be used for example to:
Introduce mutations (point mutations, short indels etc) into endogenous loci
Epitope tag endogenous genes
Create endogenous reporter systems (eg. gene fusion products with split fluorescence proteins)
Genome Editing Core provides CRISPR knock-in services for various applications using the non-viral Cas9 RNP nucleofection method. Please contact us for your project needs!
CRISPR Genome Engineering
We also offer consultations and/or services related to other CRISPR/Cas methods such as:
- Lentiviral library screens
- Knock-ins and base editing
- Epigenetic modification by recombinant deactivated Cas proteins
Contact Genome Editing Core staff for more information on how we can enable your imagination!