As today’s most common gene-editing system, CRISPR-Cas9 has great potential for applications in cancer treatment, genetic disease therapy, and animal and plant gene research. Although its DNA cleavage is highly efficient for targeted modification, multi-target editing, and RNA editing, CRISPR/Cas has limitations: without a PAM (Protospacer Adjacent Motif) near the target gene, editing cannot occur, and off-target effects remain a challenge.
Off-target effects are the main factor limiting the widespread application of gene-editing technology. Accurately assessing and detecting off-target effects, and developing strategies to reduce them, is a key research direction. Studies have shown that whole-genome sequencing is one of the simplest and most effective ways to detect off-target effects. In 2015, a research team at the Korean Institute of Life Sciences and Technology published an article in Nature Methods, Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells, highlighting the role of whole-genome sequencing in identifying off-target effects and introducing the Digenome-seq method.
CRISPR-Cas12f1 System and DNBSEQ-T7 Sequencing
Recently, the same South Korean team published their latest research in Nature Biotechnology, using the DNBSEQ-T7 platform* (MGI) to assess CRISPR-Cas12f1 off-target effects with Digenome-seq sequencing. By remodeling gRNAs, the researchers developed a highly specific CRISPR-Cas12f1 tool. Whole-genome sequencing confirmed the system’s high specificity and reduced off-target sites. Delivered via AAV vectors, the system enables efficient, multiplexed genome editing.
gRNA Design and Cas12f1 Optimization
Off-target effects largely depend on gRNA design. Non-specific binding between gRNAs and non-target regions increases off-target activity. The research team optimized Cas12f1 gRNAs by identifying five potential modification sites (MSs) in tracrRNA and crRNA sequences, creating a compact, powerful system. Engineered gRNAs significantly increased indel frequency at target loci, with the average efficiency of a single gRNA increasing by up to 867-fold.
Cas12f1’s compact design also allows dsDNA cleavage outside the protospacer sequence, enabling continued editing even after initial indel mutations.
Specificity Assessment
To ensure the system’s accuracy, the team used Cas-OFFinder to predict potential off-target sites. Digenome-seq—a whole-genome sequencing method based on in vitro nuclease digestion—was used to profile off-target activity. Sequencing with DNBSEQ-T7* confirmed fewer off-target sites and a lower off-target/on-target ratio compared with previous systems.
Therapeutic Potential
The team explored AAV-mediated CRISPR-Cas12f1 therapy. Using sgRNAs targeting the c.2991+1655A>G mutation, AAV delivery induced higher deletion levels in HEK293T cells. Cas12f1 showed approximately 46% higher deletion efficiency than control, demonstrating its potential as an efficient and versatile genome-editing system for therapy.
DNBSEQ-T7 Sequencing Platform
MGI’s DNBSEQ-T7* sequencer successfully completed whole-genome sequencing in this study, confirming the CRISPR-Cas12f1 system’s low off-target effects. Launched in 2018, the ultra-high-throughput DNBSEQ-T7 can sequence up to 60 human genomes per day, producing up to 6 Tb of data. This platform ensures accurate, high-quality sequencing for gene editing validation and off-target analysis.
Other MGI platforms have also contributed to CRISPR research. In 2020, the Neuroscience Research Institute at Peking University used the DNBSEQ-G50* to sequence gene-editing targets and potential off-target sites, verifying system specificity. In the same year, BGI Group, Shenzhen, developed the DocMF platform on MGI’s DNBSEQ system* for high-throughput identification of protein-DNA binding, cleavage sites, and CRISPR off-target sites.
References
[1] Kim, D.Y., et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nature Biotechnology 40, 94–102 (2022).
[2] Xianji.com: Gene editing “wherever you say,” four off-target detection methods.
