The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and offtarget cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less offtarget mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable 2 diseases caused by genetic mutation.
CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used to induce double-strand breaks in target DNA and activate the in-vivo DNA repair system for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious and often carcinogenic mutations can occur. Offtarget mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. In this study, we developed a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. We used various genome editors, including CRISPR-Cpf1, Cas9, and an adenine base editor, to induce intracellular genome mutations.The CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than did an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow to detect genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
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