Biology subjects

Arake de Tacca, L.

Publications and source records attributed to Arake de Tacca, L..

4 recordsLinked to original sources

bioRxiv2024

Viral delivery of compact CRISPR-Cas12f for in vivo gene editing applications

Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduce drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying in vivo CRISPR editing to diverse genetic sequences and organs in the body.

molecular biology↗

bioRxiv2023

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We then engineer the nuclease (termed AiEvo2) for increased specificity, PAM recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntingtons patient derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 CAR-T therapy from primary human T cells using multiplexed simultaneous editing and CAR insertion. To further ensure precise editing, we engineered an anti-CRISPR protein (ErAcr) to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel ErAcr protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

biochemistry↗

bioRxiv2023

Anti-CRISPR protein mediated degradation of Cas9 in human cells

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. Optimized expression of AcrIIA1 in human cells provided robust inhibition of SpyCas9 editing but had no impact on Type V CRIPSR-Cas12a, consistent with its binding site on the HNH domain. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

biochemistry↗

bioRxiv2023

Casμ, a novel class of miniature type-V Cas12f nucleases with diverse PAM

Small CRISPR-Cas effectors are key to developing gene editing therapies due to the packaging constraints in viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type-V Cas12f effectors present a solution given their small size. Here we describe Cas, a novel class of miniature (<490AA) type-V Cas12f nucleases that cleave double stranded DNA in human cells. We determined their optimal trans-activating RNA (tracrRNA) empirically through rational modifications which resulted in an optimal single guide RNA (sgRNA). We show that the Cas nucleases have broader PAM preferences than previously known Cas12f effectors. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.

biochemistry↗