Non-viral strategies for delivering genome editing enzymes.

Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.

Delivery of Cas13a/crRNA by self-degradable black phosphorus nanosheets to specifically inhibit Mcl-1 for breast cancer therapy.

Mcl-1 amplification has been observed in breast cancer and demonstrated as a key determinant of breast cancer cell survival. However, the clinical use of available effective Mcl-1-specific inhibitors for breast cancer treatment remains a challenge. An RNA-guided CRISPR/Cas13a system targeting RNAs can be used to specifically knock down mRNA expression in mammalian cells. The goal of this work is to develop a self-degradable nanoplatform based on polylysine (PLL)-functionalized black phosphorus (PBP) for the delivery of Cas13a/crRNA complexes to specifically inhibit Mcl-1 at transcriptional level for breast cancer therapy. The constructed Cas13a/crRNA complex is delivered into the cytoplasm by PBP via endocytosis, followed by endosomal escape based on the biodegradation of PBP, and this efficiently knocks down the specific gene at transcriptional level up to an efficiency of 58.64%. Through designing CRISPR RNA crMcl-1, Mcl-1 can be specifically knocked down at transcriptional level in breast cancer cells, resulting in the down-regulation of the expression of Mcl-1 protein and inhibition of the cell activity. Notably, PBP/Cas13a/crMcl-1 shows an excellent tumor suppression efficacy up to 65.16% after intratumoral injection. Therefore, biodegradable PBP is an ideal nanoplatform for the delivery of CRISPR/Cas13a, which could provide a potential strategy for gene therapy.

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents.

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.