Genome editing via CRISPR / Cas9 system
The use of the CRISPR / Cas9 system in mammalian cells has proven to be a very practical way to modify the cellular genome at a specific locus. It involves transient transfection into mammalian cells either:
(a) one or more plasmids encoding Cas9, a specific gRNA, and optionally a sequence to be inserted; or
(b) a mixture of one or two plasmids and an RNA molecule (gRNA).
Check out the CRISPR-Cas9 Webinar: an overview, challenges and the latest technologies.
Genome editing using the CRISPR / Cas9 system
The combination of CRISPER (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPER-associated nucleases (Cas) is an innovative technology for generating gene knockouts or for introducing defined sequence modifications or deletions in the genome. The CRISPR / Cas9 system is a prokaryotic immune mechanism that confers resistance to foreign genetic elements and consists of a DNA nuclease called Cas9 and a non-coding guide RNA (gRNA). The gRNA directs the Cas9 nuclease to a specific complementary locus. The nuclease subsequently induces a double-strand break.
When using the CRISPR / Cas9 system, transfection is often a key and limiting step in ensuring successful genome editing. There are three main approaches to gRNA transfection and Cas9 protein expression. It is a system of DNA, RNA or RNP delivery (gRNA + Cas9 protein). Each system has its pros and cons in terms of ease of use, genome editing efficiency, and off-target effects. The introduction of gRNA and Cas9 in the form of a plasmid is a very efficient system for easily transfected cell lines, but it is not so suitable for difficult-to-transfect primary cells and cancer cell lines. Another parameter to consider is the time for which Cas9 is to be expressed in cells, as non-specific nuclease activity may occur. DNA-free systems in which the Cas9 protein is introduced as mRNA, or better yet, as a protein, can improve the specificity of Cas9 endonuclease activity.
Li, Y. (2016) Exploiting the CRISPR / Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions. PLoS One 11 e0144970
Shi, J. & al. (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526 660-5
D’Osualdo, A. (2015). Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress. PLoS ONE 10 e0130635
Moore, R., (2015) CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells. Nucleic Acids Res 43 1297-303
Polyplus products for genome editing via CRISPR / Cas9 system
Polyplus jetPRIME® reagent is ideal for in vitro plasmid DNA-based genome editing. It requires a small amount of DNA and is suitable for multiple transfections of the plasmid while maintaining excellent cell viability.
The jetMESSENGER® reagent is ideal for efficient cotransfection of Cas9 mRNA and gRNA. With Cas9 mRNA and gRNA, faster gene editing can be achieved because transport to the nucleus is no longer required and Cas9 transcription is no longer required. The transition from plasmid DNA transfection to Cas9 mRNA transfection allows genome editing in a number of primary cell and cell line studies.
JetCRISPR® is the latest innovative agent designed to directly introduce Cas9 as a protein along with gRNA, also known as ribonucleoprotein (RNP) delivery. Both transcription and translation to obtain a functional Cas9 protein are completely omitted, making the introduction of RNP by jetCRISPR ™ the fastest, most efficient and most accurate method of gene editing currently available for a wide variety of cells.
We recommend using the in vivo-JetPEI® reagent for in vivo genome editing. Thanks to it, you will achieve excellent transfer of nucleic acids to various organs in vivo through various systems.