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Original link:Metabiology - CRISPR/Cas9
CRISPR/Cas9 is a technique that enables precise editing of specific loci in the genome. The principle is that the endonuclease Cas9 protein recognizes specific genomic sites through guided RNA (gRNA) and cleaves double stranded DNA, causing DNA double strand breaks. Cells repair the cleavage sites using non homologous end joining (NHEJ) or homologous recombination (HR) methods, achieving DNA level gene knockout or precise editing.
The CRISPR/Cas9 system mainly consists of two parts:
1. Single stranded guide RNA (sgRNA)
2. Cas9 protein with endonuclease activity
CRISPR gene knockout
When targeting gene knockout, Cas9 sgRNA can be used to cleave DNA to produce double stranded breaks. Subsequently, cells repair DNA through non homologous end connections. During this process, random deletions or additions of bases are introduced, causing frameshift mutations in the gene and resulting in the knockout of the coding gene.
CRISPR gene editing
When targeting gene insertion or replacement, the principle of homologous recombination needs to be utilized. The Cas9 sgRNA in the CRISPR/Cas9 system cleaves at the target site to produce DNA double strand breaks. In the presence of template DNA, cells repair the DNA through homologous recombination, and template DNA can be artificially designed as a gene that needs to be inserted or repaired. At this time, cells encode the target gene.
Selection of carriers (partial)
Provide virus vectors for single and double loading systems containing Cas9 protein, which can design multiple gRNAs based on the target gene sequence. The vectors carry multiple fluorescent resistance markers for easy screening.
Carrier category | number | Carrier element |
CRISPR lentivirus single vector | H5070 | pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3xFLAG-spCas9-WPRE |
H7072 | pLenti-U6-spgRNA v2.0-CMV-BSR-P2A-3xFLAG-spCas9-WPRE | |
H6825 | pLenti-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9-WPRE | |
CRISPR lentivirus dual vector | H5068 | pLenti-U6-spgRNA v2.0-CMV-EGFP-WPRE |
H5450 | pLenti-CMV-Puro-P2A-3xFLAG-espCas9_1.1-WPRE | |
CRISPR AAV Single Carrier System | H6941 | pAAV-CMV-SaCas9-U6-sagRNA v2.0 |
H5273 | pAAV-hSyn-SaCas9-U6-sagRNA v2.0 | |
CRISPR AAV Dual Carrier System | H6291 | pAAV-CMV-EGFP-WPRE-U6-spgRNA v2.0 |
H12663 | pAAV-U6-shRNA/spgRNA v2.0-CMV-EGFP-WPRE | |
H11012 | pAAV-CMV-Luc2-WPRE-U6-spgRNA |
Virus Carrier Service Process
