Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Authors:
Farboud B1, Jarvis E2, Roth TL3, Shin J4, Corn JE4, Marson A5, Meyer BJ1, Patel NH6, Hochstrasser ML7.
In:
Source: J Vis Exp
Publication Date: (2018)
Issue: 2018: 135
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
T cell, human cord blood unstim.
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofectorâ„¢ X-Unit
Experiment

CRISPR / Cas9 RNP Nucleofection with the 4D Nucleofector. Mobilized human CD34 cells from Lonza (AllCells 4Y-101C, 3Y-101D) and C. elegans are used as models in this video to show how fast and easy genome editing can be done with best practize tips and troubleshooting guidelines. See the video here: https://www.jove.com/video/57350/enhanced-genome-editing-with-cas9-ribonucleoprotein-diverse-cells


Abstract

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.