Methods of improving target specificity of genome editing through the improvement of CRISPR systems and the use of new gene editing tools. (A) Method for improving target specificity by CRISPR-Cas9 protein engineering. Top: In the case of eSpCas9, the (+) charged residue that binds to the non-target strand of the target DNA is replaced with a neutrally charged amino acid (K848A, K1003A, R1060A). Bottom: In the case of SpCas9-HF1, the (+) charged residue that binds to the target strand of the target DNA is replaced with neutrally charged amino acids (N497A, R661A, Q695A, Q926A). (B) A method for improving target specificity by guide RNA engineering of CRISPR-Cas effectors. In the chimeric RNA-DNA that binds to the target DNA, blue region in protospacer represents RNA and red region represents DNA, respectively. (C) Method for improving target specificity using Prime editor. Depending on the PE2, PE3, and PE3b methods, different efficiencies and types of prime editing were induced on the target DNA. (D) A family tree of new types of gene editing tools (TnpB, Fanzor). Blue region indicates PBS + RTT sequence of sense DNA and pink boxed region indicates the protospacer of second guide RNA for anti-sense DNA nick generation, respectively. RTase: Reverse transcriptase, pegRNA: prime editing guide RNA. Schematic showing evolution from TnpB with the simplest functional domain to the CRISPR-Cas12 (Top) and the Fanzor (Bottom) effectors, respectively. Images were created with BioRender.com.
