FLI-Seq™ amplifies ONLY the CRISPR insert, avoiding PCR duplications
While performing CRISPR screens, researchers face challenges with the PCR amplification step before sequencing. The large amount of DNA used in published protocols inhibits the PCR reaction. To get enough DNA for sequencing, scientists have to increase the number of PCR cycles. Increased PCR cycles can be problematic when amplifying a library, as the final results are usually PCR duplicates or artifacts with no “useful” information.
FLI-Seq™ solves this problem by removing undesired genomic DNA while enriching for the region of interest. In the case of CRISPR screens it will enrich the DNA fragment containing the single guide RNA (sgRNA).
Once enriched, this fragment can be easily amplified with less PCR cycles, avoiding PCR duplications and obtaining robust and reproducible results.
FLI-Seq™ improves Library Prep for CRISPR pooled screens by recovering the majority of sgRNAs
FLI-Seq™ removes 99.8% of genomic DNA while keeping sgRNA yields
Figure 1. Bars indicate enrichment performed on biological replicate Day 10 samples (A = ~4.4×106cells, B = ~6.9×106cells). Shown are input gDNA (dsDNA), supernatant (ssDNA), and pulldown (ssDNA) yields quantified by Nanodrop 2000 .
FLI-Seq™ yields are near equivalent to genomic DNA sgRNA yields
Figure 2. Points indicate (y-axis) yield (quantified by high-sensitivity D1000 tapestation) after 20 cycles of PCR amplification starting with (x-axis) indicated input gDNA amounts (or equivalent sample fraction for purified and supernatant samples) .
Highly reproducible method at 35×-50× coverage = 5× more CRISPR Screens
Figure 3. Schematic of infection and technical replicates to query experimental reproducibility. Coverage was extrapolated based on gDNA yield using standard 6.6 𝜇g per 1 million cell estimates .
Figure 4. Heatmap indicates correlation (Pearson R) between reads-per-million normalized sgRNA counts (log2) from technical or biological replicate samples as indicated, using only sgRNAs with RPM>1 in at least one dataset .
FLI-Seq™ produces robust next generation sequencing CRISPR library preps while decreasing the technical variability.
Large-scale screening has long been a popular tool in the life sciences community to identify functional regulators of a phenotype of interest in an unbiased, genome-wide manner. In particular, CRISPR screening has become a powerful method to characterize thousands of different genes in a single experiment .
Standard CRISPR/Cas9 generates a population of cells representing individual knockouts across the entire genome while avoiding multiple knockouts occurring in the same cell [3, 4]. Cells are then subjected to some sort of selective pressure or treatment (Figure 5), followed by genomic DNA isolation, library preparation and high-throughput sequencing.
Figure 5. Selection of CRISPR pooled screens
Typical library preparation from CRISPR pooled screens involves standard genomic DNA isolation followed by PCR amplification [3, 5]. However, this simple description belies the challenge of amplifying the library without introducing bias into the relative target frequencies.
A variety of methods have been described to alleviate this concern, as simply as performing multiple (often 30 or more) PCR reactions per sample or performing restriction digestion and size-selection to remove unrelated genomic DNA .
However, due to the complexity and inefficiency of these methods (at large scale), there is need for a new approach that is able to amplify the fragment of interest while discarding non useful material, producing robust libraries with high reproducibility.
FLI-seq uses biotin-coupled RNA oligonucleotides that show near-complete recovery of desired sgRNA target regions while removing unwanted genomic DNA background. This allows low-amplification library preparation at scale and with decreased technical variability.
Eric L. Van Nostrand, Sarah A. Barnhill, Alexander A. Shishkin, David A. Nelles, Eric Byeon, Thai Nguyen, Yiu Chueng Eric Wong, Nathan C. Gianneschi, Gene W. Yeo. Unbiased identification of nanoparticle cell uptake mechanism via a genome-wide CRISPR/Cas9 knockout screen. doi: https://doi.org/10.1101/2020.10.08.332510
Doench JG. Am I ready for CRISPR? A user’s guide to genetic screens. Nature reviews Genetics. 2018;19(2):67-80. doi: 10.1038/nrg.2017.97. PubMed PMID: 29199283.
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-7. doi: 10.1126/science.1247005. PubMed PMID: 24336571; PubMed Central PMCID: PMC4089965.
Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343(6166):80-4. doi: 10.1126/science.1246981. PubMed PMID: 24336569; PubMed Central PMCID: PMC3972032.
Kampmann M, Bassik MC, Weissman JS. Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps. Nature protocols. 2014;9(8):1825-47. doi: 10.1038/nprot.2014.103. PubMed PMID: 24992097; PubMed Central PMCID: PMC4144868.