Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively.

Functionally diverse type V CRISPR-Cas systems.

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i.

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr.

Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation.

The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus.

CRISPR-Cas systems are small RNA-based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR-Cas systems including representatives from the three major types of CRISPR-Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non-coding RNAs) associated with the respective locus.

Binding and cleavage of CRISPR RNA by Cas6.

The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids.

Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes.

An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses.