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Liu, W.-H. ; Völse, K. ; Senft, D. ; Jeremias, I.

A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models.

Sci. Rep. 11:12649 (2021)

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CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

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