A brief introduction of CRISPR gene editing system
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) is a bacterial defense system that 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 asingleguide 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.
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Cas9 nuclease can form ribonucleoprotein (RNP) complex with the single guide RNA (sgRNA) component of the CRISPR/Cas9 system, inducing site-specific DNA double-stranded breaks. The PAM sequence is NGG.
When LwCas13a recognizes and cleaves the target RNA under the guidance of guide RNA, its "accessory cleavage" activity is activated, which can efficiently cleave non-specific single-stranded RNA (ssRNA) in the reaction system.
SpRYCas9 is an RNA-mediated nuclease, which can catalyze the specific site cleavage of double-stranded DNA. It can recognize the NNN (NRN > NYN) PAM sequence, which almost gets rid of the restriction of the PAM.
LbaCas12a (Cpf1) has a RuvC endonuclease domain similar to Cas9 but does not have the HNH endonuclease domain. LbaCas12a is not only smaller than Cas9, but also requires a smaller RNA (nearly half of Cas9's total sgRNA), which is very helpful for genome editing.
Compared with other Cas proteins, the molecular weight of Cas14 protein is generally smaller (400-700aa). Similar to Cas12, Cas14a1 can also bind the target nucleic acid and activate its ssDNA trans cleavage activity.
The 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.
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.
The sgRNA is directly synthesized via a chemical approach. Research shows that synthetic sgRNA has more consistent editing efficiencies and lower off-target effects compared with plasmid and IVT sgRNAs.