Arrayed lentiviral CRISPR screening? Not so fast. Lessons learned from RNAi

Considerations before using arrayed lentiviral single guide RNA libraries

Whole genome arrayed shRNA libraries were described starting early in 2004^1-3 and lentiviral libraries have been commercially available to researchers for over a decade [reviewed in⁴]; and yet only two whole genome screens using these resources have been published (Table 1). Given the enthusiasm for adopting CRISPR-based approaches, let’s take a look at the known limiting factors in using arrayed lentiviral resources, and what we can learn from this work as CRISPR arrayed screening advances as a discovery method.

Lentiviral particles are not conducive to high-throughput arrayed screening

While lentiviral shRNA reagents have been extremely useful in both small-scale knockdown experiments and large-scale pooled library screens, very few truly high- throughput arrayed screens have been performed and published (Table 1). A review of published arrayed shRNA screens reveals several critical (and potentially limiting) success factors. Clear guidelines for handling lentiviral particles have been established from this body of work, and compliance with these guidelines makes it impractical to work at genome-scale without putting the project at high risk for experimental variability.

Publication	# shRNA # genes Plate format	Titer MOI	Special handling to improve transduction or Control for infection efficiency	Cell handling
Maier et al.2009	2,660 905 96-well	NA NA 100 µL	Filtered lentivirus before transduction	Medium change at 1 day
Barbie et al.2009	5,002 957 384-well	NA NA NA	Quadruplicate plates with duplicate plates treated with puromycin. Ratio of puromycin- positive to puromycin-negative values was used to assess infection efficiency	Medium change at 2 days – for duplicates
Scholl et al. 2009	5,024 1,011 384-well	NA ~ 5 NA	Quadruplicate plates with duplicate plates treated with puromycin. Ratio of puromycin- positive to puromycin-negative values was used to assess infection efficiency	Medium change at 2 days – for duplicates
Colombi et al. 2011	80,000 14,308 96-well	> 1 × 106 TU/mL ~ 1 30 µL	No special handling reported	After 1 day – split cells into 2 plates
Delmore et al. 2011	5,024 1,011 384-well	NA NA NA	Quadruplicate plates with duplicate plates treated with puromycin. Ratio of puromycin-to assess infection efficiency	Medium change at 2 days – for duplicates
Bhinder et al. 2013	80,598 16,039 384-well	> 1 × 106 TU/mL MOI up to 5 4 µL	Lentiviral particles thawed at room temperature and spun for 1 min on benchtop. Then lentivirus added and spun for 8 min on benchtop	Medium changes at 3 days and 7 days

Table 1. Summary of published arrayed lentiviral shRNA screens
NA = not available

Consistency of titer:
It is not practical to measure the actual functional titer of every well of a lentiviral particle library, so commercial providers frequently provide the titer as a minimum specification. For example, at least 1 × 106 TU/mL^5,6. Even when stored at -80 °C, unpurified lentiviral particles will lose ~10x in titer every 6-12 months. A single freeze-thaw of supernatant, low titer lentiviral particles can reduce titer by 2- to 3-fold. Unknown titers will impact transduction efficiency, which is key to success in an arrayed screen. One method for improving transduction efficiency is centrifuging plates after lentiviral particles are added to the cells⁶, but this is not feasible for a large screen of hundreds of plates.
Safe handling:
Lentiviral particles must be thawed on ice just prior to use, and require appropriate containment. This is impractical for many labs when considering a whole genome consists of hundreds of microtiter plates. If the solution is to divide up the plates and perform the screen across many different days, then variability is inevitably introduced.
Crude supernatant: Because lentiviral particle concentration methods, such as ultracentrifugation and column purification, are not amenable to high-throughput implementation, researchers often use the crude supernatant. Supernatant contains cell debris that can cause cellular toxicity, so filtration may be necessary if you are adding high volumes of lentiviral particles to transduce your cells as was the case for Maier et al.⁷.
Extra handling required for antibiotic selection:
Experimental protocols for lentiviral transduction require several liquid handling steps. All the publications listed in Table 1 carried out at least one medium change for removing transduction medium (containing cell debris and excess non-transducing lentiviral particles) and adding antibiotic-containing medium for selection^{2, 5- 10}. In addition, antibiotic selection and longer time points also required cell splitting in some cases⁵. Splitting cells from multi-well plates without introducing variability and contamination, while complying with safety regulations, is technically challenging, and higher cell density at longer time points prevents the application of high-content analysis of cell phenotypes.

Lessons learned from shRNA screens are applicable to sgRNA screens

Maier et al. took advantage of the unique quality of a one-gene-per-well format using an shRNA lentiviral library targeting 905 genes. They applied a sophisticated assay where circadian phenotypes were measured over several days using a luciferase reporter⁷. This work demonstrated that with smaller libraries and a robust assay, lentiviral arrayed screening is feasible.

The current generation of arrayed lentiviral sgRNA libraries was first described in 2015, but the use of these reagents in gene knockout was demonstrated for only a few genes¹¹. One arrayed lentiviral sgRNA screen has been published to date; McCleland et al. described using an arrayed lentiviral sgRNA library with 1,030 sgRNA constructs targeting 200 genes¹². From the methods, we deduced that, like arrayed shRNA screens, titer and transduction efficiency are still challenges to consider. These researchers employed centrifugation for 30 minutes to improve transduction, and calculated the ratio of cell death from puromycin-selected vs. unselected cells to determine transduction efficiency. Finally, viability was measured as a phenotype.

Clearly, arrayed lentiviral sgRNA for gene knockout is a valuable resource for experimentation. Almost 2,000 peer-reviewed publications have used smaller numbers of individual lentiviral sgRNA constructs to target genes for knockout. Using these smaller scale experiments, complex phenotypes have been observed. Using lentiviral sgRNA constructs in arrayed format for screening has potential as long as lentiviral handling guidelines and experimental requirements such as medium changes and selection can be applied in a high-throughput setting.

Recommended alternatives to arrayed lentiviral sgRNA libraries

Alternative sgRNA library formats such as arrayed synthetic oligos and lentiviral pools provide screeners the same experimental opportunities, without the complications mentioned above. Available coverage includes the whole genome, pre-designed gene families, and also custom Cherry-pick for just your favorite genes. For more information, and to see which is best for your needs, please see this article.

Additional Resources

Edit-R Arrayed Lentiviral sgRNA Libraries
Arrayed collections of lentiviral sgRNA libraries for high-throughput knockout screening across entire human gene families.
Cherry-pick lentiviral sgRNA libraries
Order custom libraries of predesigned lentiviral sgRNA constructs as arrayed glycerol stocks
Edit-R Pooled Lentiviral sgRNA Libraries
High-titer pooled screening libraries for pre-defined gene sets in human and mouse

References

J. Moffat et al., A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283-1298 (2006).
K. Berns et al., A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431-437 (2004).
P. J. Paddison et al., A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427-431 (2004).
Q. Pan, L. J. van der Laan, H. L. Janssen, M. P. Peppelenbosch, A dynamic perspective of RNAi library development. Trends Biotechnol 30, 206-215 (2012).
M. Colombi et al., Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction. Oncogene 30, 1551-1565 (2011).
B. Bhinder et al., An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates. Assay Drug Dev Technol 11, 173-190 (2013).
B. Maier et al., A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes Dev 23, 708-718 (2009).
D. A. Barbie et al., Systematic RNA interference reveals that oncogenic KRAS- driven cancers require TBK1. Nature 462, 108-112 (2009).
J. E. Delmore et al., BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904-917 (2011).
C. Scholl et al., Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 137, 821-834 (2009).
T. Schmidt, J. L. Schmid-Burgk, V. Hornung, Synthesis of an arrayed sgRNA library targeting the human genome. Sci Rep 5, 14987 (2015).
M. L. McCleland et al., CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer. The Journal of Clinical Investigation 126, 639-652 (2016).