Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells

Madsen RR, Semple RK
Publication Date: (2019)
Issue: 11;4: 37
Research Area:
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

Preparation of CRISPR/Cas9 targeting reagents: Single-stranded oligo DNA (ssODN) templates, ALT-R XT CRISPR RNA (crRNA), ALT-R trans-activating crRNA (tracrRNA) and ALT-R Cas9 Nuclease V3 were acquired from Integrated DNA Technologies (IDT).

Prior to use, the crRNA and the tracRNA were resuspended in pH 7.5 TE buffer (IDT # 11-01-02-02) to a final concentration of 100 µM. To prepare crRNA:tracRNA duplexes at 50 µM, equal volumes of each RNA were mixed and heated for 5 minutes at 95 °C, followed by controlled cool-off to 25 °C at ramp rate 0.1 °C/second. The formed duplexes were placed on ice until ready to use.

Ribonucleoproteins were prepared by mixing 5 µl each of crRNA:tracRNA duplex (50 µM) and recombinant Cas9 enzyme (61 µM), followed by incubation at room temperature for 20 minutes. Next, 200 pmol of each HDR template was added to the RNPs prior to delivery into iPSCs.

Nucleofection and luminescence-based screening of CRISPR-targeted iPSCs: Targeting was performed using healthy, subconfluent iPSCs (P51) pretreated with 1X RevitaCell (in E8/F) for 3 hours.The cells were dissociated with StemPro Accutase (Thermo Fisher Scientific # A110501) and 1e6 cells taken forward for nucleofection. Following low-speed centrifugation (100G, 3 minutes), the cell pellet was resuspended in 100 µl nucleofection solution (P3 Nucleofection Kit, Lonza # V4XP-3024). Of this suspension, 85 µl were transferred to the assembled RNPs, resulting in c. 850,000 cells in the final reaction. The nucleofection was carried out with an Amaxa 4D nucleofector, using programme CA137. Immediatley following nucleofection, 500 µl E8/F with 1X RevitaCell were added to the cell suspension and transferred to a Falcon tube containing 4.5 ml E8/F with 1X RevitaCell.


Human pluripotent stem cells are increasingly used for CRISPR-mediated gene targeting in efforts to generate models of human diseases. This is a challenging task because of the high sensitivity of these cells to suboptimal conditions, including CRISPR-associated DNA damage and subsequent rounds of single-cell cloning. We sought to develop a sensitive method that enables rapid screening of CRISPR targeted cells, while preserving cell viability and eliminating the need for expensive sequencing of a large number of clones. A protocol was designed in which the luminescent peptide tag, HiBiT, is appended to the extracellular portion of an inert surface membrane protein (CD46), using synthetic CRISPR reagents and a widely distributed human induced pluripotent stem cell (iPSC) line. We find that this approach substantially reduces labour-intensive screening of CRISPR-targeted iPSCs and minimises the number of subcloning steps. Successfully edited iPSCs could be identified within a week of targeting, based only on extracellular luminescence detection in live cells. The total screening time in each round was less than 30 minutes and no sequencing was required. This method can be developed further to serve as a highly sensitive co-selection strategy in CRISPR knock-in experiments, particularly in the context of challenging cell lines.