I intend to do CRISPR KO. Can I insert gRNA into lentiCRISPR V2 via infusion cloning? Also, will the whole sequence of lentiCRISPR V2 be incorporated into host genome or only partially? Where would the insertion be? Wouldn't consistent CRISPR expression be problematic? What are the advantages of CRISPR KO compared to shRNA? Thanks in advance!
Yes, you can use in-fusion cloning with lentiCRISPR V2 along with the standard digestion and ligation method. According to Takara, the minimal size of the fragment for in-fusion cloning is 50bp, which contains 15bp left and right overlapping sequence and 20bp insert.
Depends on how you use the plasmid, you could use it for transient transfection and expression without worrying about integration. Partial integration will happen if you do long term antibiotics selection or deliver the plasmid as lentivirus.
Consistent CRISPR expression should be avoided when possible, as it increases the chance of off-target effect and may be toxic to the cell. However, many CRISPR screening projects requires consistent CRISPR expression.
With proper design, CRISPR KO can be more specific and efficient than shRNA.
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