RESUMEN
Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.
RESUMEN
Clinical genome-wide next-generation sequencing (NGS) has brought new challenges to genetic laboratories. The identification of numerous patient-specific variants that may require to be screened for on multiple other samples poses an issue when striving for time and cost-effectiveness. Here, we propose d-multiSeq, a straightforward method utilizing the advantages of droplet PCR for multiplexing combined with amplicon-based NGS. By comparing d-multiSeq with a standard multiplex amplicon-based NGS, it was shown that partitioning prevents the amplification competition seen when multiplexing and leads to a homogeneous representation of each target in the total read count for up to a 40-target multiplex without the need for prior adjustment. Variant allele frequency was reliably evaluated with a sensitivity of 97.6% for variant allele frequency up to 1%. The applicability of d-multiSeq was also tested on cell-free DNA with the successful amplification of an eight-target multiplex panel. Preliminary application of the technique to assess the clonal evolution in a childhood leukemia harboring high interpatient variability in its somatic variants is shown. d-multiSeq represents a turnkey solution for analyzing large sets of patient-specific variants on low DNA amounts and cell-free DNA.
