Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

Abstract

This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1–Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma

“…To this end, we used our recent protocol 16 to quantify the frequency of indel mutations generated by the cellular DNA repair machinery as a measure of the cutting efficiency of our CRISPR/Cas9 constructs (Supplementary Figure 1 A ). Next, we co-transfected the various construct pairs into Neuro-2a cells and assessed their ability to generate the Dnajb1–Prkaca fusion three days post-transfection.…”

“…Neuro-2a cells were transfected with the individual pX330 gRNA/Cas9 constructs along with empty pSpCas9(BB)-2A-GFP vector 15 at a 6:1 molecular ratio to mark transfected cells with GFP, using X-tremeGENE HP transfection reagent according to the manufacturer’s instructions (Roche). Two days post-transfection, we analyzed the efficiency of the gRNA designs using our protocol for genome editing using FACS enrichment of nuclease expressing cells and indel detection by amplicon analysis (IDAA) 16 . Briefly, the top-10% most GFP fluorescent cells were isolated by FACS and lysed to 2000 cells/μl in QuickExtract DNA extraction solution (Epicentre).…”

Tandem paired nicking promotes precise genome editing with scarce interference by p53

“…IDAA IDAA was performed as described previously (Carrington et al, 2015;Lonowski et al, 2017;Yang et al, 2015). Briefly, 293T cells were transfected with PX459-, PX462-or pH840Apuro-based constructs designed for PIGA or CD55, and gDNAs were extracted -17 -after three days of incubation.…”

“Therapeutic applications of the ‘NPGP’ family of viral 2As”

“…89 Regardless of the nuclease system used, it is essential to have methods that can select or enrich genome-modified cells. Several approaches using 2A have been applied to aid selective enrichment of transfected cells, including fusion of ZFNs/TALENs and Cas9 to a fluorescent or antibiotic protein, [97][98][99][100][101] or, co-transfection of the nucleases with a fluorescent or antibiotic resistance marker gene. 100 To recapitulate DNA nuclease activity, dual-reporter surrogate systems that contain the target sequence to be modified have been developed to detect cells with high repair efficiency.…”