Question: Why does my CRISPR Screening library trace have additional peaks?
Answer: We expect to observe a narrow peak at ~300bp in CRISPR Screening libraries generated using our Single Cell 3’ Gene Expression and CRISPR Screening protocol (standard throughput and HT) and ~270bp using our 5’ CRISPR protocols. Unlike 3’ CRISPR, 5’ CRISPR does not require capture sequence integration and therefore libraries may be closer in size to ~270bp. The example shown below was generated from sgRNA constructs described in our Guide RNA Specifications Compatible with Feature Barcoding technology for CRISPR Screening (CG000197), but can vary depending on the sgRNA construct selected by the user and whether the guide has a capture sequence integrated.
Additional peaks in 200-500 bp size range
In some cases, additional peaks may be observed in the size range of ~200bp to ~500bp. These peaks are off-target fragments that arise during PCR amplification in cases where sgRNA abundance is low.
If CRISPR libraries with off-target peaks at ~200-500bp are sequenced, the “Fraction Guide Reads” will be lower than expected, given that the off-target library fragments will not map to sgRNA sequences. To improve metrics in future experiments, several troubleshooting avenues to consider can help increase sgRNA abundance and sgRNA capture efficiency in the 10x assay, as outlined below.
1) Selection strategy for sgRNA expression
To ensure that a high percentage of cells express sgRNAs, the following optimization strategies can be considered:
Increase the number of days of antibiotic selection
- If the sgRNA vector expresses an antibiotic resistance gene, antibiotic selection can be performed to enrich sgRNA-expressing cells.
- Increasing the number of days of antibiotic selection may improve performance in the 10x CRISPR Screening assay. During in-house experiments, we typically perform at least 7 days of antibiotic selection post sgRNA-transduction.
Use a two-step selection strategy (antibiotics + FACS)
- If using a sgRNA vector that also expresses a fluorescent reporter, a two-step strategy can be used to increase the percentage of sgRNA-expressing cells and improve performance in the 10x CRISPR Screening assay.
- Cells can be selected with antibiotics and also sorted based on fluorescence. This two-step selection strategy has been demonstrated in Replogle et al. 2020
2) Selection strategy for Cas9 expression
We observed reduced sgRNA capture efficiency in the 10x assay during in-house experiments when using cells without Cas9/dCas9 expression. We hypothesize that sgRNAs are protected from nuclease digestion when bound by Cas9/dCas9. Therefore, sgRNA abundance is decreased in the absence of Cas9/dCas9, which results in low sgRNA capture efficiency in the 10x assay.
To ensure that your cell population has robust expression of Cas9/dCas9, the following optimization strategies can be considered:
Maintain antibiotics in the media throughout the experiment
- If antibiotics are used to select for Cas9/dCas9 expression, we recommend maintaining the antibiotics in the media throughout the duration of the experiment to ensure that cells do not lose Cas9/dCas9
- For in-house experiments, we typically use blasticidin selection to generate cells with stable expression of dCas9-KRAB. We then maintain blasticidin in the media during subsequent sgRNA transduction and selection.
Use a fluorescent reporter to sort for Cas9-expressing cells
- In some cases, such as when working with primary cells, it may not be possible to generate stable cell lines with Cas9/dCas9 expression.
- A Cas9/dCas9 vector with a fluorescent reporter could be used in these situations so that FACS can be performed to select cells with robust Cas9/dCas9 expression.
3) Capture Sequence and Integration Site
The choice of Capture Sequences and Integration Sites can impact sgRNA capture efficiency in the 10x CRISPR Screening assay. See: Which Capture Sequence and sgRNA integration site should I use for CRISPR Screening experiments?
4) Vector design
It is important to use sgRNA vectors that are robustly expressed in your cell type. We recommend purchasing lentiviral pools from MilliporeSigma, as their lentiviral vectors have been validated with the 10x CRISPR Screening assay in a variety of cell types.
5) MOI for sgRNA transduction
In some cases, we have found that increasing the MOI used for sgRNA transduction can improve performance in the 10x CRISPR Screening assay. It may be beneficial to perform an MOI titration to find the optimal MOI that leads to efficient sgRNA transduction but does not result in too many cells expressing more than one sgRNA.
Note that it is typically not problematic if a subset of cells expresses more than one sgRNA. These cells can be identified in Cell Ranger and excluded from the final analysis.
6) Do not modify the CRISPR library preparation protocol in the 10x User Guide
If off-target peaks at ~200-500bp are observed, we do not recommend attempting to reduce these off-target peaks by modifying the 10x CRISPR User Guide's protocol steps. During internal product development, this protocol's steps were extensively optimized to increase on-target and decrease off-target fragments. Additional modifications, such as increasing PCR cycle number, increasing PCR input volumes, or modifying SPRIselect ratios, are unlikely to improve assay performance. Instead, we recommend optimizing upstream steps (e.g., sgRNA transduction & selection) to increase sgRNA abundance in your cell sample.
Note on additional peaks greater than 1 kb.
In some cases, higher molecular weight peaks (>1kb) may be present. These peaks are not problematic and do not negatively affect sequencing or assay performance.
Product: Single Cell Gene Expression, Single Cell Immune Profiling