Introduction

The CRISPR/Cas9 system has emerged as a revolutionary tool for genome editing, enabling researchers to precisely modify genes of interest. However, achieving high knockout efficiency remains a challenge, especially in patient-derived organoid models. These models resemble in vivo conditions more accurately but are not well amenable to lipofection. In this study, we propose an innovative strategy to enhance the knockout efficiency of CRISPR/Cas9 ribonucleoproteins (RNPs) by co-targeting hypoxanthine-guanine phosphoribosyltransferase (HPRT), a key enzyme in the purine salvage pathway and subsequently treating with 6-thioguanine. Here, we demonstrate that this co-knockout strategy can significantly increase knockout efficiency of a gene of interest in patient-derived colorectal cancer (CRC) organoids.

Aims & Methods

HPRT is an enzyme involved in the salvage pathway of purine nucleotide synthesis. The anticancer agent, 6-thioguanine, relies on HPRT to be metabolized into cytotoxic 6-thioguanine nucleotides. Subsequent incorporation of these nucleotides into DNA and RNA mediates its cytotoxic effect. It has been previously shown in 2D cell lines that the functional significance of HPRT in the purine salvage pathway can be exploited to enhance the knockout efficiency of a gene of interest using CRISPR/Cas9: by co-targeting HPRT alongside the gene of interest, HPRT-deficient cells are unable to metabolize 6-thioguanine and thus become resistant to its cytotoxic effects. Consequently, 6-thioguanine can be used as a selective agent, enabling the enrichment of cells with HPRT KO together with the desired knockout. We have successfully redesigned and optimized this method for use in CRC organoids.

Results

We have established an easy-to-scale 384-well CRISPR/Cas9 RNP transfection platform to generate high-efficiency gene knockouts (KO) in adherent cell lines. Depending on the individual sgRNA used, we were able to achieve a genome editing efficiency of 40-60 percent 48 hours after transfection. We show that this editing efficiency can be further increased by co-targeting HPRT with CRISPR RNP transfection and subsequently treating with 6-thioguanine. In the effort to extend this approach to CRC organoids, we optimized electroporation conditions with CRISPR RNPs and sgRNA produced via in vitro transcription. We demonstrate high knockout efficiencies of more than 40 percent in FACS analysis after knockout of surface markers CD81, CD46 & CD151 with two sgRNAs, respectively. In Sanger sequencing genotyping (analysis with Synthego ICE CRISPR analysis tool), we were able to achieve knockout efficiencies of 20 percent at individual guide target sites.

Conclusion

Here, we report an innovative method for generating high-efficiency CRISPR/Cas9 RNP knockout pools in patient-derived CRC organoids. We intend to apply this method to verify and further investigate the knockout effects of specific genes of interest identified in arrayed CRISPR RNP screens conducted using our 384-well RNP transfection platform in adherent CRC cell lines. Furthermore, our method is particularly valuable for generating knockout organoids without the need for lentiviral transduction. Of note, CRISPR RNPs offer the advantage of quicker editing and lower off-target rates when compared with DNA-based CRISPR delivery methods. High-efficiency knockouts in patient-derived organoids without the need for lentiviral transduction are essential for developing new targeted therapies for colorectal cancer.