Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells

Abstract

Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

“…Although a few strategies for Cas9-RNP delivery have been reported, these strategies suffer from endosomal entrapment of both Cas9 protein and sgRNA (Liu et al , 2015). Mechanical methods including membrane deformation (Han et al ., 2015), electroporation (Schumann et al ., 2015), and the use of hypertonic agents (D’Astolfo et al ., 2015) provide direct delivery, however, they require specialized instrumentations and are generally not practical for in vivo therapeutic applications.…”

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…This strategy has been successfully used to perform genome editing in mouse inner ear hair cells and in neurons Wang et al, 2016). An additional advantage of the direct Cas9:sgRNA protein:RNA complex delivery over mRNA or DNA delivery is the more transient nature of protein delivery, which results in substantially higher DNA specificity and less off-target editing, for the reasons discussed above Zuris et al, 2015;Liu et al, 2015b).…”

“…This strategy improves genome-editing specificity as it reduces the amount of time Cas9 can function after its ontarget locus has already been modified and only off-target loci are available for modification. For example, the direct delivery of Cas9:sgRNA ribonucleotide protein complexes (RNPs) to cells, which results in transient Cas9 activity, rather than plasmid transfection, which results in long-lasting Cas9 and sgRNA expression, can increase the ratio of on-target genome editing to off-target genome editing by more than an order of magnitude in mammalian cells (Lin et al, 2014b;Kim et al, 2014;Ramakrishna et al, 2014;Zuris et al, 2015;Liu et al, 2015b).…”

Genome-Editing Technologies: Principles and Applications

“…Off-target cleavage has also been reduced by controlling the dosage of either the Cas9 protein or gRNA within the cell , or even by using Cas9 variants configured to enable conditional genome editing, such as a rapamycininducible split-Cas9 architecture (Zetsche et al 2015b) or a Cas9 variant that contains a strategically placed small-molecule-responsive intein domain . Nucleofection or transient transfection ) of a preformed Cas9 ribonucleoprotein complex has also been shown to reduce offtarget effects, enabling DNA-free gene editing in primary human T cells (Schumann et al 2015), embryonic stem cells (Liu et al 2015b), Caenorhabditis elegans gonads (Paix et al 2015), mouse (Menoret et al 2015;Wang et al 2015a) and zebrafish embryos , and even plant protoplasts (Woo et al 2015). The incorporation of specific chemical modifications known to protect RNA from nuclease degradation and stabilize secondary structure can further enhance Cas9 ribonucleoprotein activity (Hendel et al 2015;Rahdar et al 2015).…”