What is CRISPR?
A brief introduction of CRISPR gene editing system
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) is a bacterial defense system which forms the basis for CRISPR/Cas gene editing technology. This new gene editing technology is now becoming a more efficient and customizable alternative to other existing tools.
CRISPR system contains two components: a CRISPR-associated endonuclease (Cas nuclease) and a single guide RNA (sgRNA). Cas protein snips through DNA like a pair of molecular scissors, and sgRNA directs Cas protein to a specific site of DNA to make the cut. The protospacer adjacent motif (PAM) is a short DNA sequence following the target cutting site. The PAM is also a prerequisite for a Cas nuclease to cut.
sgRNAs
A programmable GPS from different sources
1st GenerationThe sgRNA sequence is first cloned into a plasmid vector, and then introduced into cells by transfection, which is suitable for High-Throughput Gene Editing. This is the most original method, which requires more than a week for the preparation before the gene editing experiment.
2nd Generation
The sgRNA is first transcribed from a DNA template by RNA polymerase. Additional purification is then required before the experiment. Generally, making an In Vitro-transcribed (IVT) sgRNA takes around 3 days.
3rd Generation
The sgRNA is directly synthesized via chemical approach. Research shows that synthetic sgRNA has more consistant editing efficiencies and lower off-target effects compared with plasmid and IVT sgRNAs.
