Abstract 2772: Comparison of three single-cell CRISPR-Seq protocols on the 10X Genomics platform

Different methodologies exist for single-cell CRISPR (scCRISPR) screens, each with its own requirements concerning sgRNA scaffold, sgRNA library vector design, instrumentation, and reagents. This study compares the performance of three distinct scCRISPR protocols: 10X Genomics 3’ scCRISPR, CROP-Seq (using a modified 10X 3’ scCRISPR protocol), and 10X Genomics 5’ scCRISPR. 10X 3’ scCRISPR relies on the CS1 capture sequence inserted in the sgRNA tracr to detect expressed sgRNAs. CROP-Seq protocol requires the sgRNA sequences to be embedded in an expressed mRNA for detection. 10X Genomics 5’ scCRISPR detects sgRNAs without the need for scaffold modifications or inclusion in a mRNA transcript. A modification of the 10X 5’ scCRISPR protocol also allowed us to incorporate sc-targeted RNA-seq, decreasing NGS costs and potentially leading to better transcriptional profiling data. The findings of the study showed that while the CROP-Seq and 10X-CS1 approaches are equally effective for single-cell sgRNA detection and gene expression profiling, the CROP-seq approach was superior in the sgRNA’s ability to induce the expected phenotype. The 10X 5’ scCRISPR protocol proved to be superior to both CROP-Seq and 10X-CS1 in sgRNA detection, and comparable if not better to CROP-seq in gene expression profiling and sgRNA activity. Also, the 5’ scCRISPR targeted RNAseq approach proved to be technically successful, although it did not provide the expected transcriptional profiling improvement. Given the ease of use, the quality of the results, and the lack of need for specialized sgRNA scaffold or library design, 10X 5’ scCRISPR should be the protocol of choice for scCRISPR screens utilizing the 10X Genomics platform. Citation Format: Donato Tedesco, Tianbing Liu, Mikhail Makhanov, Nadya Isachenko, Dongfang Hu, Paul Diehl, Alex Chenchik. Comparison of three single-cell CRISPR-Seq protocols on the 10X Genomics platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2772.