To address this, EDITGENE has compiled and answered some of the most frequently encountered issues, aiming to help researchers design more robust experimental strategies and accelerate their project timelines.

No. To save time, many researchers prefer to purchase ready-to-use products, such as pre-amplified and quality-controlled library plasmids/viruses, or cell pools already transduced with sgRNA libraries. If the plasmid or viral library quality is reliable (meeting coverage and uniformity standards), this can indeed accelerate your experimental progress. However, we do not recommend using cell pools already transduced with sgRNA libraries directly for screening.

The main reason is that cells are typically delivered frozen, so they must be recovered before experimentation. Differences in gene function and regulatory mechanisms mean that gene knockouts can affect cell viability to varying degrees. During recovery, cells carrying different gene edits may differ in survival and proliferation rates, which can lead to the loss of certain sgRNAs. This, in turn, can severely compromise library coverage and uniformity, making the quality of the library unreliable. Using such a cell pool for downstream functional screening would therefore lack scientific validity.

It is generally not recommended to use primary cells. When selecting a cell line for CRISPR library screening, the following criteria should be considered: 1) compatibility with the research objective, 2) correct species origin, 3) ability to undergo stable passaging, and 4) high transfection efficiency. Common choices include tumor cell lines, immortalized cell lines, and stem cells/iPSCs.

Primary cells are freshly isolated and cultured immediately from tissues, which allows them to more accurately mimic the in vivo environment. This makes them appealing for library screening experiments. However, most primary cells are terminally differentiated and have limited proliferation and passaging capacity. During library construction, this often leads to difficulties in expanding the cell population, preventing the generation of a sufficiently large cell pool for subsequent selection. Additionally, loss of certain sgRNAs can reduce library coverage and uniformity.

If primary cells must be used, it is advisable to work with smaller libraries (fewer sgRNAs) and lower cell coverage, and to proceed with selection experiments as soon as the cell pool is established.

1) Is it necessary to use a single-clone Cas9 stable cell line?
In theory, single-clone lines are preferred because they ensure stable and uniform Cas9 editing efficiency. However, based on our experience and published reports, using a polyclonal Cas9 stable cell line for library screening generally does not significantly affect the accuracy of screening results.

2) How to improve Cas9 editing efficiency in stable cell lines?
Within certain limits, higher Cas9 expression can enhance editing efficiency. For example, when transducing cells with Cas9 lentivirus, you can increase the MOI as much as possible without compromising cell viability. This increases the Cas9 copy number and overall expression level, thereby improving editing efficiency.

3) How to assess Cas9 editing efficiency?
A common first approach is to measure Cas9 expression levels, but this only provides an indirect indication. For a more comprehensive evaluation, we recommend using validated high-efficiency sgRNAs, delivering them to the Cas9 stable cell line, and then confirming editing efficiency via sequencing.

When calculating the required amount of plasmid library, it must be sufficient to support subsequent selection experiments. Multiple factors should be considered, including the size of the sgRNA library, cell MOI, cell coverage, and the number of groups in the selection experiment. Losses at each experimental step—such as during plasmid amplification, large-scale plasmid prep, viral packaging, and cell transduction—also need to be accounted for. Therefore, it is recommended to amplify extra plasmid to ensure sufficient supply.

Based on our experience, determining the necessary plasmid quantity involves calculating the transformation efficiency during plasmid amplification. By counting colonies after a defined dilution, the total number of clones can be estimated. The total clone number should reach 500× to 1000× the number of sgRNAs to meet experimental requirements. If the clone number is insufficient, additional electroporation rounds should be performed to achieve adequate library coverage.

Plasmid library amplification is typically performed using electroporation or chemical transformation; using a nucleofector is not recommended.

Electroporation is currently the most efficient method for plasmid transformation. In this approach, plasmid DNA is mixed with electrocompetent cells, and an electric pulse is applied to facilitate DNA entry. After electroporation, cells are plated on selective media to isolate colonies carrying the desired plasmid. High-efficiency competent cells are critical for success.

Chemical transformation is generally not suitable for large plasmid libraries, as its lower efficiency can compromise library uniformity and coverage after amplification. For smaller plasmid libraries, chemical transformation can be attempted, but electroporation remains the preferred method for maintaining library quality.

1) Confirm how the viral titer is measured
Currently, viral titers are often determined using qPCR. However, this method does not accurately reflect the titer of infectious virus, because qPCR detects viral genome fragments from both live and inactivated virus. In our experience, the actual infectious titer is typically only about one-tenth of the value measured by qPCR. Therefore, we recommend performing a direct cell-based titration to determine the effective viral titer.

2) Use a titration experiment to establish the real experimental system for transduction
In practice, take a small amount of library virus and prepare a series of dilutions. Transduce the target cell line with each dilution while recording all experimental parameters, including viral dose, cell number, selection drug concentration, and culture conditions. From this pre-experiment, select the condition that achieves roughly 30% transduction efficiency. When performing the actual transduction, scale these conditions proportionally to match the desired experiment size.

Points to note:

1) gRNA extraction:
Pay careful attention to the number of cells in each experimental group. The total cell number should be 400-1000× the number of sgRNAs in the library, so that each sgRNA is represented by roughly 400–1000 cells. This ensures sufficient coverage for reliable sequencing. Additionally, avoid overloading DNA purification columns, as this can reduce the diversity of sgRNAs in the sample.

2) gDNA PCR:
Nested PCR (two rounds of amplification) can be used to increase specificity for target fragments. Keep the number of PCR cycles as low as possible to minimize bias.

Amplification issues:

If sequences fail to amplify, it may be necessary to re-evaluate PCR conditions, such as primer concentration, cycle number, and reaction system. For Illumina sequencing, using the correct primers and sequencing strategy is crucial to accurately capture sgRNA sequences. If amplification still fails, check the library construction process and quality to ensure that sgRNA sequences were correctly inserted into the library.
Every step in the experiment involves many critical details, and overlooking them can affect the success of the entire CRISPR library screening. This brief guide aims to highlight key points and provide practical advice to help optimize your experiments.

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