Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a 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 off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a 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–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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In 2012, it was discovered that precise gene editing could be induced in the target DNA using the reprogrammable characteristics of the CRISPR system. Since then, several studies have investigated the potential of the CRISPR system to edit various biological organisms. For the typical CRISPR system obtained from bacteria and archaea, many application studies have been conducted and have spread to various fields. To date, orthologs with various characteristics other than CRISPR-Cas9 have been discovered and are being intensively studied in the field of gene editing. CRISPR-Cas12 and its varied orthologs are representative examples of genome editing tools and have superior properties in terms of in vivo target gene editing compared to Cas9. Recently, TnpB and Fanzor of the OMEGA (obligate mobile-element guided activity) system are identified to be the ancestor of CRISPR-Cas12 based on phylogenetic analysis. Notably, the compact sizes of Cas12 and OMEGA endonucleases allows adenosine-associated virus (AAV) delivery, hence, they are set to challenge Cas9 for in vivo gene therapy. This review will focus on these RNA-guided reprogrammable endonucleases: their structure, biochemistry, off-target effects, and applications in therapeutic gene editing.

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.