Gene knockout Cloning

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Gene knockout Cloning

·    Technical Details    ·
        CRISPR/Cas9 system is a defense mechanism used by bacteria to resist the invasion of viruses and foreign plasmids. At present, the type II CRISPR/Cas9 system is the most developed and widely used system. It recognizes the target sequence with gRNA, and guide Cas9 endonuclease to cut the upstream of PAM, resulting in the double-strand break (DSB) of the target site DNA. To repair the DSB, the cell uses its own DNA repair mechanism to add or delete or replace pieces of DNA sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).
·    Gene knockout strategies   ·
Strategy 1
Short fragment removal. Guide RNAs target introns at both sides of exon 2 and the number of bases in exon 2 is not a multiple of 3, which can cause frame-shift mutation.
Strategy 2
Frame-shift mutation. Guide RNA targets the exon, and the base number of deletion is not a multiple of 3. After knockout, frame-shift mutation would cause gene knockout.
Strategy 3
Large fragment removal. Complete removal of the coding sequence to achieve gene knockout.
·   Classification of knockout gRNA plasmids  ·
Ubigene developed a series of YKO plasmids, including lentivirus plasmids, AAV plasmids, and non-viral plasmids, which can be widely used in vitro or in vivo.
YKO series Vector Reporter gene; Selection marker
Lentiviral YKO plasmid YKO-LV001-single-gRNA
YKO-LV001-dual-gRNA
EGFP/mCherry,Puro/Neo
EGFP/mCherry,Puro/Neo
Non-viral YKO plasmid YKO-RP001-single-gRNA
YKO-RP001-dual-gRNA
EGFP/mCherry,Puro/Neo
EGFP/mCherry,Puro/Neo
AAV YKO plasmid YKO-AAV001-single-gRNA
YKO-AAV001-dual-gRNA
EGFP/mCherry
EGFP/mCherry
Single  gRNA
plasmid can be used in frame-shift mutation or short fragment knockout. By co-transfecting 2-3 gRNAs, knockout efficiency can be improved.
Dual  gRNA
Dual-gRNA plasmid can be used in large fragment removal. Dual-gRNA can greatly improve the efficiency of cell transfection and gene knockout.
Validation
Restriction enzyme digestion and sequencing
Application
Different species: mammals, zebrafish, primary cells, stem cells, cell lines, microorganisms, etc.
Different purposes: frame-shift mutation knockout, fragment knockout, precise knockout, point mutation, fragment knockin.

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