Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNA-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9.

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes.

Genome editing using RNA-guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5'-NNGRRT-3', compared with Streptococcus pyogenes Cas9 (SpCas9) of 5'-NGG-3' PAM. Here, SaCas9-RNP-mediated genome editing is reported in human cells.

Basic and Clinical Application of Adeno-Associated Virus-Mediated Genome Editing.

Traditional gene therapy (gene replacement) has made a breakthrough in treating inherited diseases. Adeno-associated virus (AAV) has emerged as a highly promising vector with innate ability, boosting the development of gene replacement and gene targeting. With the recent advance of engineered nucleases that work efficiently in human cells, AAV mediated-genome editing with nucleases has raised hopes for gene therapy of inherited and non-inherited diseases.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells.