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.

Cell-free protein synthesis of CRISPR ribonucleoproteins (RNP).

Cell-free protein synthesis is a powerful tool to produce recombinant proteins, and as an open system, can often integrate all or part of downstream assays. Here we describe in vitro synthesis of the Streptococcus pyogenes type II-A CRISPR-Cas9 ribonucleoproteins (SpCas9 RNPs), consisting of the effector protein and the single guide RNAs (sgRNAs). In spite of its large molecular weight (160kDa), the SpCas9 effector is expressed relatively well from linear DNA templates under T7 promoter in commercial reconstituted cell-free protein synthesis systems.

Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array.