CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1,...CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.展开更多
基金National Natural Science Foundation of China(Grant No.31571403)the Natural Science Foundation of Beijing Municipality(Grant No.2171001)
文摘CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.
