Background
CRISPR-Cas systems are widely used in prokaryotes, where they provide adaptive immunity to viral infection and plasmid transformation. Over the past decade, RNA-programmable CRISPR-Cas nucleases have emerged as powerful tools for efficient and precise genome editing in living cells, and it has revolutionized biotechnology. Cas nucleases with trans-cleavage activity have been widely used in nucleic acid detection, especially Cas12a. Cas12a has both dsDNA cleavage activity and target activated ssDNA byactivity (trans-cleavage activity). However, Cas12a's large size and low specificity for single-base mismatch recognition hinder its application in this field.
The off-target and unwanted editing effects that are permanently incorporated into target cells are the main worries of researchers working on CRISPR detection systems or therapeutics. Off-target detection screens have shown that predicting locations that are vulnerable to mistaken CRISPR cleavage is difficult and is influenced by the gRNA sequence, tissue or cell type, the specific nuclease employed, and the delivery technique. Off-target activity is counterbalanced by effective editing. The goal of therapeutic editing is, of course, to have a high on-target to low off-target ratio.
CRISPR-CasΦ (CRISPR-Cas12j), a RNA-directed enzyme found in phages, is about half the size of Cas9 and Cas12a, but retains the ability to unpack and cleft dsDNA, making it valuable in gene editing. Compared with Cas12a, CasΦ has better targeting specificity, but its cleavage activity is lower than that of Cas12a. Therefore, based on a rational design strategy and the introduction of hairpin structured crRNA, we hope to construct a new CRISPR-Cas system with high cleavage activity and good target recognition specificity.
The off-target and unwanted editing effects that are permanently incorporated into target cells are the main worries of researchers working on CRISPR detection systems or therapeutics. Off-target detection screens have shown that predicting locations that are vulnerable to mistaken CRISPR cleavage is difficult and is influenced by the gRNA sequence, tissue or cell type, the specific nuclease employed, and the delivery technique. Off-target activity is counterbalanced by effective editing. The goal of therapeutic editing is, of course, to have a high on-target to low off-target ratio.
CRISPR-CasΦ (CRISPR-Cas12j), a RNA-directed enzyme found in phages, is about half the size of Cas9 and Cas12a, but retains the ability to unpack and cleft dsDNA, making it valuable in gene editing. Compared with Cas12a, CasΦ has better targeting specificity, but its cleavage activity is lower than that of Cas12a. Therefore, based on a rational design strategy and the introduction of hairpin structured crRNA, we hope to construct a new CRISPR-Cas system with high cleavage activity and good target recognition specificity.
