For CRISPR knockout in HeLa cells, please recommend Cas9 protein and sgRNA kits.

For membrane protein genes, it is recommended to design at least two sgRNAs to avoid cell compensation after single-site cleavage or difficulties in amplifying the target region. Additionally, include a positive control targeting a known locus, such as AAVS1 or HPRT, to determine whether the issue lies in gRNA design or the delivery workflow. Harvest gDNA 48-72 h after cleavage for T7E1 or PCR-Sanger sequencing; do not rely solely on cell phenotype. For HeLa electroporation, the cell density should not be too high; start with 0.5×10^5 to 1×10^5 cells per well in a 24-well plate, use the lower limit of RNP amount according to the instructions, and monitor cell viability. For membrane protein genes with high GC content, avoid repeat regions when designing sgRNAs.

When comparing suppliers, I look at four dimensions: Cas9 activity calibration data, sgRNA purity and modifications, delivery lead time, and whether small samples are provided. After receiving the goods, it is best to first perform a cleavage assay using the positive substrate included in the kit or a control gRNA to rule out transport and storage issues. Some domestic suppliers can provide Cas9 activity premixed substrate, allowing you to test within a few days whether it is usable. When purchasing, do not focus only on unit price; sgRNA degradation will directly cause editing failure. After arrival, aliquot by use and store at -80°C, and modified sgRNAs should also avoid repeated freeze-thaw cycles. After membrane protein knockout, it is recommended to simultaneously perform flow cytometry or Western blot to determine protein disappearance, rather than relying solely on genomic sequencing.

When the budget is limited, I buy Cas9 protein and sgRNA separately, which makes it easier to control costs. For synthetic sgRNA, I have the supplier perform HPLC purification; a 2 nmol specification is enough for dozens of operations in a 24-well plate. For Cas9 protein, buy small packages of 50-100 pmol; do not start by buying large specifications. In terms of delivery speed, domestic brands such as Nearshore, General Biosystems, and Tsingke are usually faster than imported ones, and many have items in stock, so an order placed today can arrive the day after tomorrow. Imported brands may have better batch-to-batch consistency, but lead times are long. Ask clearly whether the sgRNA has end modifications and whether the Cas9 has endotoxin testing data, especially since membrane protein knockout is sensitive to HeLa cell status.

When we first did HeLa knockout, we also used plasmids, and at 30% transfection the editing-positive rate was indeed low. Later we switched to preforming RNP from Cas9 protein and chemically synthesized sgRNA, then delivering it by electroporation with Lonza or Neon, bypassing the plasmid transcription and translation steps, which gave a more concentrated cleavage window. For Cas9, you can choose NEB, Thermo, or domestic small-pack options from Nearshore/GenScript; for sgRNA, choose the 2'-O-methyl-modified type, which is more stable against degradation. For 4-6 wells in a 24-well plate, it is recommended to first test 2-3 electroporation conditions with one target and not use everything up at once. Within a budget of 3000, most small-size combinations are sufficient; first request a small sample or the smallest pack to verify activity.