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 engineer the nuclease (termed AiEvo2) for increased specificity, protospacer adjacent motif 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 Huntington's 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 chimeric antigen receptor-T-cell therapy from primary human T cells using multiplexed simultaneous editing and chimeric antigen receptor insertion. To further ensure precise editing, we engineered an anti-CRISPR protein to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel engineered anti-CRISPR protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

PTBP1 mRNA isoforms and regulation of their translation.

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells and identified alternative 5' and 3' untranslated regions (5'-UTRs, 3'-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.

Viral Delivery of Compact CRISPR-Cas12f for 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 reduces 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 CRISPR editing to diverse genetic sequences and organs in the body.

Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.

Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of 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. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. 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.