1. What is PRCISR™?

PRCISR™ is Vivlion’s CRISPR library and screening technology platform. PRCISR™ libraries are produced using Vivlion’s proprietary 3Cs technology, a PCR-free and cloning-free approach designed to generate highly uniform CRISPR sgRNA libraries.

3Cs is the underlying library production technology, while PRCISR™ is the broader platform encompassing Vivlion’s CRISPR library formats, screening solutions, and related services.

2. What is Vivlion’s 3Cs technology?

3Cs stands for Covalently-Closed-Circular-synthesis. It is Vivlion’s proprietary technology for producing CRISPR libraries using a PCR-free and cloning-free library production workflow.

3. How does 3Cs technology work?

The 3Cs process uses an oligonucleotide pool directly in the library synthesis reaction rather than first amplifying the pool by PCR. The oligo pool is annealed to a ssDNA and extended to generate a dsDNA plasmid. The resulting DNA is subsequently amplified in bacteria to generate the final CRISPR library.

4. How does 3Cs technology compare with conventional approaches?

Conventional CRISPR library production typically involves PCR amplification and cloning of an oligonucleotide pool before generating the final library. Each of these steps can introduce sequence-dependent amplification bias or cloning artifacts that affect sgRNA representation.

The 3Cs approach avoids PCR amplification and conventional restriction/ligation cloning of the oligonucleotide pool. By reducing these production steps, 3Cs is designed to better preserve the representation of the original pool and generate highly uniform CRISPR libraries.

3Cs supports the annealing of multiple oligonucleotide pools onto a single ssDNA template, rather than being limited to a single oligonucleotide pool. This capability allows the generation of complex dual-targeting CRISPR libraries.

1. Which CRISPR modalities and library types does Vivlion offer?

Vivlion offers CRISPR knockout (CRISPRko) libraries as part of its off-the-shelf library portfolio, as well as custom CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) libraries.

Vivlion’s current library portfolio is compatible with the Cas9 enzyme. Vivlion can also provide custom library designs for other CRISPR-associated enzymes, including Cas12a and Cas13d, depending on your experimental requirements.

Vivlion specializes in pooled libraries but arrayed CRISPR libraries for validation can be assembled upon request.

To discuss a custom CRISPR library for a specific Cas enzyme or application, contact our science team at info@vivlion.com.

2. Why are uniformity and sgRNA representation important?

Uniform sgRNA representation is important for ensuring that individual perturbations are adequately represented throughout a CRISPR screen. If some sgRNAs are strongly overrepresented while others are present at very low abundance, underrepresented perturbations may have insufficient coverage and their biological effects may be harder to detect, potentially masking true hits.

Vivlion’s library production approach is designed to maintain a uniform sgRNA distribution and minimize representation bias. This helps ensure broad and consistent coverage of the library, supporting reliable identification of screening hits while potentially reducing the experimental scale required to achieve sufficient representation of individual perturbations.

3. How does Vivlion select sgRNAs for a CRISPR library?

Vivlion combines publicly available CRISPR screening data, computational prediction, and experimental validation to identify high-performing sgRNAs and generate a genome-wide sgRNA database.

sgRNA selection considers key factors such as predicted on-target activity and potential off-target effects, helping prioritize sgRNAs that are expected to efficiently and specifically perturb their intended targets.

This data-driven approach helps Vivlion select sgRNAs that provide effective and specific target perturbation, while supporting consistent performance across CRISPR screens.

4. How many sgRNAs are typically used per gene?

The number of sgRNAs per gene depends on the library. For example, Vivlion’s Alexandria single-targeting human library uses four unique sgRNAs per gene.

5. What controls are included in a CRISPR library?

The appropriate controls depend on the CRISPR screening strategy and experimental readout. A well-designed library may include several types of controls to help assess background effects, perturbation efficiency, and assay performance. These controls help distinguish true biological effects from background or technical variation and support the interpretation and reproducibility of screening results.

For example, Vivlion’s Alexandria fixed-pair human library includes non-targeting controls, reporter/marker controls, and safe-harbor controls. Custom libraries may also include other neutral, depletion, or enrichment controls tailored to specific experimental purposes.

1. What CRISPR library backbones are available at Vivlion?

Vivlion offers a range of validated PRCISR™ CRISPR backbones for pooled CRISPR screening libraries. Vivlion’s backbone toolbox supports different library formats, including single-targeting, fixed-pair, and multiplex libraries.

Backbones can be configured with different selection markers, fluorescent reporters, and other functional modules depending on the requirements of the screening experiment.

2. Can I use my own backbone for CRISPR library production?

Yes, Vivlion can work with customer-provided backbones for customized CRISPR library projects. We can assess the backbone design and compatibility with the intended sgRNA architecture, selection system, and library format before production. This allows existing customer vectors to be incorporated into the PCR-free 3Cs library workflow.

3. Can I customize a CRISPR library backbone for my needs?

Yes, Vivlion’s PRCISR™ CRISPR backbones can be customized for specific screening requirements. Depending on the application, customization can include elements such as selection markers, fluorescent reporters, barcodes, and other vector components. Vivlion can also help determine the appropriate backbone configuration based on the cell type, screening strategy, and intended readout.

4. Can the nuclease and sgRNA be expressed from the same plasmid?

Yes, depending on the experimental requirements, Vivlion can design CRISPR vectors that combine nuclease and sgRNA expression on the same plasmid.

Alternatively, and more commonly, the nuclease and sgRNA can be delivered as two separate plasmids. The preferred configuration depends on the cell type, delivery method, screening format, and experimental objective.

5. Which selection markers can I use for my CRISPR library?

Vivlion can incorporate different selection markers into customized CRISPR library backbones, depending on the experimental setup and vector configuration. Puromycin, blasticidin, and neomycin are used in several Vivlion CRISPR library configurations, while other selection systems can be considered for customized projects.

6. What fluorescent reporters can be added to my CRISPR library?

Fluorescent reporters can be incorporated into customized PRCISR™ CRISPR library backbones when a fluorescent readout or enrichment strategy is required.

Common reporter options include mCherry, EGFP, BFP, RFP. Other fluorescent proteins can also be incorporated depending on the experimental design and compatibility with the cell line and assay. Vivlion’s off-the-shelf Alexandria libraries are also available with mCherry.

7. Can I customize the tracrRNA in my CRISPR library?

Yes, Vivlion can customize the tracrRNA configuration of a PRCISR™ CRISPR library when required by the experimental setup or nuclease used.

For our standard library designs, we use benchmark-tested tracrRNA configurations that have been validated as part of our library development and screening workflows. This helps ensure reliable sgRNA expression and consistent library performance.

If your project requires a specific tracrRNA or a different sgRNA architecture, we can evaluate the requested configuration and incorporate it into a customized library where appropriate.

1. When should I use a single-targeting CRISPR library?

A single-targeting library is the standard choice when each cell should receive a single sgRNA targeting one gene.

This format is well suited for:

• • Genome-wide CRISPR knockout (CRISPRko) screens
  • Target identification
  • Essentiality screens
  • Drug-resistance or drug-sensitization screens
  • Reporter-based screens
  • Validation of gene function

Vivlion offers various genome-wide single-targeting CRISPR knockout (CRISPRko) libraries as well as customized single-targeting libraries.

2. When should I use a fixed-pair CRISPR library?

A fixed-pair library is useful when you want to deliver two predefined sgRNAs to the same cell. These sgRNAs can either target the same gene or two different genes.

This format is particularly useful for applications such as:

• • Combinatorial gene editing
  • Paralog or gene-family studies
  • Genetic interaction studies
  • Synthetic lethality screens
  • Synthetic rescue screens
  • Targeted DNA excision
  • Gene activation/upregulation screens

Vivlion’s fixed-pair PRCISR™ CRISPR libraries maintain predefined sgRNA combinations while preserving high library uniformity.

3. When should I use a multiplex CRISPR library?

A multiplex library is appropriate when the experiment requires two sgRNAs to be delivered to the same cell. The sgRNAs are randomly paired in every possible combination.

Vivlion’s multiplex libraries are designed for combinatorial CRISPR screening and can be used to investigate genetic interactions, synthetic vulnerabilities, and functional relationships between genes.

Multiplex libraries are particularly useful when the goal is to explore a large combinatorial search space rather than test predefined pairs.

4. When should I use a CROP-seq library?

A CROP-seq library is a good choice when you want to combine pooled CRISPR perturbations with single-cell RNA sequencing. It links the CRISPR perturbation in each cell to the transcriptomic profile, allowing to study how individual genetic perturbations affect cell states and gene-expression programs.

Vivlion supports the CRISPR library design and screening part of the experimental pipeline and collaborates with Single Cell Discoveries to support customers with downstream single-cell RNA sequencing workflows such as single-cell barcoding, sequencing, and data analysis.

Together, the workflow can cover the process from CROP-seq library design and CRISPR screening through single-cell RNA sequencing and downstream analysis. 

5. When should I use a minimized genome-wide CRISPR library?

A minimized genome-wide library can be useful when reducing library complexity is important, for example when cell numbers, screening capacity, or experimental scale are limiting factors.

A standard genome-wide library provides broader sgRNA representation and redundancy, while a minimized library uses fewer sgRNAs per gene to reduce overall library size.

Vivlion’s Pergamon library is designed as a minimized genome-wide CRISPR library, while Alexandria provides higher sgRNA redundancy. The appropriate choice depends on the desired coverage, available cell numbers, and screening objectives.

6. How do PRCISR™ CRISPR libraries perform when cell numbers are limited?

Highly uniform CRISPR libraries can help reduce the number of cells required to maintain adequate sgRNA representation during pooled screening. Vivlion’,s data indicate that certain screening designs can be performed at substantially reduced scale, with reductions of up to approximately 10-fold reported in specific experimental contexts.

This can be particularly useful when working with scarce or difficult-to-expand cell populations, such as primary cells or iPSCs. The appropriate screening scale depends on the library, experimental design, and biological system.

1. What is the Alexandria genome-wide CRISPR library?

Named after the ancient library of Alexandria, Alexandria is Vivlion’s flagship genome-wide PRCISR™ CRISPR library, designed to provide broad genome coverage with validated, high-performing sgRNAs. Alexandria has 4 sgRNAs per gene, and it is available in both single-targeting and fixed-pair formats, supporting a range of genome-wide CRISPRko screening applications.

2. What is the Pergamon genome-wide CRISPR library?

Named after the ancient library of Pergamon, Pergamon represents a minimized approach to genome-wide CRISPR screening.

Pergamon is Vivlion’s minimized genome-wide PRCISR™ CRISPR library with 2 sgRNAs per gene, designed to provide broad genome-wide gene coverage while reducing overall library complexity. It is particularly suited for screens where maintaining genome-wide coverage is important, while reducing the required cell numbers, sequencing depth, and overall screening scale.

3. Which organisms can Vivlion CRISPR libraries target?

Vivlion can design and produce CRISPR libraries for a range of organisms, depending on the availability and suitability of target sequences and the requirements of the project.

Our existing genome-wide library portfolio includes human and mouse CRISPR knockout (CRISPRko) libraries, while customized libraries can be designed for other organisms and experimental systems.

1. In which formats can I order PRCISR™ CRISPR libraries?

PRCISR™ CRISPR libraries can be ordered in plasmid DNA or lentiviral format, depending on your experimental setup.

Plasmid DNA libraries are suitable for customers who want to perform their own downstream viral packaging or use the library directly with an appropriate delivery system.

Lentiviral libraries are available for customers who want to use the CRISPR library directly for lentiviral delivery into their cells.

2. What quality control steps does Vivlion perform for PRCISR™ CRISPR libraries?

Quality control is an integral part of Vivlion’s CRISPR library production. Vivlion applies multiple quality-control steps during CRISPR library production to assess library identity, completeness, and sgRNA representation.

Depending on the library, quality control can include:

• • Sanger sequencing

  • Full-plasmid sequencing

  • Restriction digest analysis

  • NGS-based library characterization

  • Assessment of library completeness

  • Evaluation of library uniformity and representation

A CoA is generated and provided with every library.

3. Can I use my own sgRNA sequences for PRCISR™ CRISPR library production?

Yes, Vivlion can work with customer-provided sgRNA sequences for customized CRISPR libraries.

Customer-provided sgRNAs can be assessed for factors such as on-target activity, potential off-target sites, sequence quality, and problematic sequence features before library production.

Vivlion can also help identify and remove unsuitable sequences as part of the library design process.

4. Can Vivlion design sgRNAs for my target genes?

Yes, if you provide a list of target genes, Vivlion can design and select suitable sgRNAs for your CRISPR library.

Vivlion has an established and continuously expanding sgRNA database, containing sgRNAs that have been carefully selected and validated for their targeting performance. Our sgRNA selection process is designed to maximize targeting efficiency and reliable gene perturbation while considering factors such as on-target activity and potential off-target effects.

When suitable validated sgRNAs are already available, Vivlion can use these established sequences for your library. For new targets, we can design and evaluate additional sgRNAs according to the requirements of your project and the selected CRISPR nuclease.

5. How do I prepare Vivlion CRISPR libraries for NGS?

Vivlion has established NGS sample-processing and PCR protocols for its PRCISR™ CRISPR libraries and can assist clients with those steps.

6. Can I validate my CRISPR screen hits using the Vivlion backbone?

Yes, Vivlion can provide the empty backbone for restriction-ligation cloning, allowing you to clone individual sgRNAs or custom constructs for follow-up validation experiments.

This can be useful for validating CRISPR screen hits in a specific cell line or experimental setup using the same vector architecture as the screening library. 

1. Does Vivlion offer CRISPR screening as a service?

Yes, Vivlion offers customized, end-to-end CRISPR screening services, supporting projects from experimental design and cell engineering through CRISPR library production, screening, NGS, and bioinformatic analysis. Vivlion can also support independent stages of the workflow.

2. What is included in the PRCISR™ CRISPR screening project?

The scope of a PRCISR™ CRISPR screening project is tailored to the experimental requirements. Depending on the project, services can include:

• • Experimental design and screening strategy

  • Cell engineering and preparation

  • Custom CRISPR library design and production

  • CRISPR screening

  • NGS sample preparation and sequencing

  • NGS data processing and quality control

  • sgRNA read counting and screen analysis

  • Hit identification and prioritization

  • Functional and pathway analysis

  • Biological interpretation and project reporting

Vivlion can therefore support projects from experimental planning through screening, NGS, and bioinformatics, with the exact scope and deliverables defined during project planning.

3. Can I outsource only part of a CRISPR screening project to Vivlion?

Yes, Vivlion offers modular CRISPR screening services, so customers can outsource individual parts of a project without outsourcing the complete screening workflow.

Depending on the project requirements, individual services can include:

• • Cell engineering

  • CRISPR screening

  • gDNA extraction

  • NGS sample preparation and NGS-PCR

  • CRISPR screening data analysis and bioinformatics

This allows customers to perform parts of the workflow in-house while outsourcing specific steps to Vivlion based on their technical requirements, available capacity and project needs. The exact scope and sample requirements are defined together with the Vivlion scientific team.

1. Can Vivlion design a CRISPR screening project around my research question?

Yes, Vivlion can design custom CRISPR screening projects around a specific biological question, cell system, treatment, or phenotype.

The project design can include selection of the appropriate CRISPR screening format, experimental controls, CRISPR library design, cell engineering, screening conditions, NGS strategy, and downstream bioinformatic analysis.

Vivlion can also help determine whether a single-targeting, fixed-pair, multiplex, or CROP-seq approach is most appropriate for the research question.

2. What information does Vivlion need to plan a CRISPR screening project?

To design an appropriate CRISPR screening strategy, Vivlion typically considers:

• • • Cell type and cellular system
    • Biological question or target pathway
    • Treatment, compound, or experimental condition
    • Desired screening readout or phenotype
    • Available cell numbers and experimental scale
    • Desired CRISPR library format and target genes
    • Controls and validation strategy
    • Screening duration and selection conditions
    • NGS and data-analysis requirements

The initial project discussion helps Vivlion define the appropriate CRISPR library, screening design, NGS workflow, and bioinformatics strategy for the project. 

3. Can Vivlion help select the appropriate CRISPR library for my research question?

Yes, Vivlion can help select the CRISPR library format and screening strategy that best fits your biological question and experimental setup.

The choice depends on factors such as the biological question, desired genetic resolution, cell numbers, screening scale, and readout. Vivlion can help determine the appropriate library design and screening strategy during project planning.

4. Can Vivlion perform a CRISPR screen using my existing library?

Yes, Vivlion can perform CRISPR screening using customer-provided CRISPR libraries, provided that the library is compatible with the planned screening workflow.

Before starting the project, Vivlion will review key library characteristics, such as the library format, backbone, sgRNA design, selection markers, delivery method, and available quality-control information, to confirm compatibility with the planned screen.

This allows customers to use an existing CRISPR library while leveraging Vivlion’s expertise in screening, NGS, and bioinformatic analysis

5. How long does a typical CRISPR screening project take?

The timeline for a CRISPR screening project depends on factors such as cell engineering, library production, experimental design, screening duration, sequencing, and data analysis. Vivlion’s scientific team works with customers to define a project-specific timeline based on the scope and requirements of each screen.

1. Does Vivlion provide NGS sample preparation for CRISPR screens?

Yes, Vivlion provides NGS sample preparation as a stand-alone service for CRISPR screens, including projects where the CRISPR screen has been performed by the customer.

For example, customers can send cell pellets or genomic DNA to Vivlion for downstream NGS processing. Depending on the project, the workflow can include:

• • Genomic DNA (gDNA) isolation

  • NGS-PCR amplification of CRISPR sgRNA sequences

  • NGS sample preparation and quality control

  • Sequencing

  • NGS data processing and sgRNA read counting

The exact sample requirements and NGS workflow are defined based on the CRISPR library and screening design.

2. Does Vivlion provide bioinformatic analysis of CRISPR screening data?

Yes, Vivlion provides bioinformatic analysis for CRISPR screening and NGS data, including analysis of both single-targeting and combinatorial CRISPR screens.

Depending on the project, analysis can include sgRNA read counting, quality control, enrichment and depletion analysis, hit identification, genetic-interaction analysis, functional enrichment and data visualization.

Vivlion can use established analysis tools such as MAGeCK, PinAPL-Py, BAGEL, drugZ, MAUDE, and GEMINI, as well as customized analysis workflows tailored to the screening design and biological question.

3. What does Vivlion’s CRISPR screening data analysis include?

The analysis workflow is tailored to the screening format and experimental design. Depending on the project, PRCISR™ CRISPR screening data analysis can include:

• • NGS read processing and sgRNA read counting

  • Library and sample quality control

  • Replicate correlation and control performance

  • sgRNA and gene-level enrichment/depletion analysis

  • Hit identification

  • Combinatorial gene-effect and genetic-interaction analysis

  • Functional and pathway enrichment

  • GO analysis and network biology

  • Data visualization and reporting

4. What deliverables will I receive from a Vivlion CRISPR screening project?

Project deliverables are defined according to the agreed scope of the CRISPR screening project.

Depending on the workflow, deliverables can include processed NGS data, library and screening quality control metrics, sgRNA or gene-level count tables, statistical analysis, hit lists, enrichment/depletion results, genetic-interaction analysis, functional enrichment and biological interpretation.

Vivlion defines the expected deliverables together with the customer during project planning, ensuring that the final data package addresses the objectives of the screening project. 

1. Can Vivlion provide cell engineering services?

Yes, Vivlion can provide cell engineering support for CRISPR screening projects, depending on the requirements of the experimental system such as Cas9, dCas9-VPR, and dCas9-KRAB.

Cell engineering can include establishing or modifying cell models required for the screen, introducing relevant genetic components, and preparing cells for the intended CRISPR screening workflow.

The exact engineering strategy is defined based on the cell type, delivery method, screening format, and experimental readout.

2. Can Vivlion optimize compound concentrations for drug-response CRISPR screens?

Yes, Vivlion can support compound and treatment-condition optimization as part of drug-response CRISPR screening projects.

Depending on the experimental setup, this can include testing a range of compound concentrations and treatment conditions to identify an appropriate screening window before performing the genome-wide or targeted CRISPR screen.

The optimization strategy is designed around the desired phenotype and can consider factors such as treatment duration, cellular viability and the expected dynamic range of the assay.

1. What readouts does Vivlion support for CRISPR screens?

CRISPR screens performed at Vivlion are customizable. The choice of screening readout depends on the CRISPR modality and the overall experimental question. Vivlion supports screening based on cell viability and proliferation, flow cytometry/FACS, reporter activity, and microscopy-based readouts.

2. What factors should I consider when designing a CRISPR screen?

CRISPR screen design involves considerations at two levels:

• • Biological considerations: research objectives, cell model, CRISPR modality and library, treatment conditions, and selective pressure.

  • Experimental considerations: screening scale, experimental controls, readout, screening duration, screening format, sequencing strategy, and data analysis pipeline.

At Vivlion, the scientific team first understands your scientific needs and research objectives, then works closely with you to carefully design and optimize the screening strategy.

3. How is the required cell number determined for a CRISPR screen, and can screening be performed with limited cell numbers?

The required cell number depends on the size of the CRISPR library, the desired sgRNA representation, the screening design, and the expected cell loss during transduction, selection, and the screen. Sufficient cell numbers are needed to maintain adequate representation of each perturbation throughout the experiment.

When cell numbers are limited, screening may still be feasible by using minimized or focused libraries, reducing the required representation, or optimizing the experimental design. Vivlion can help adapt the library and screening strategy to the available cell numbers while maintaining sufficient representation for meaningful results.

4. How does sgRNA representation affect screening results?

Poor sgRNA representation can cause some sgRNAs to be present at very low abundance, increasing the risk that their associated phenotypes are missed. This is particularly important in dropout screens, where low sgRNA abundance can make it difficult to distinguish true hits from phenotypes that are lost due to insufficient sgRNA representation. More uniform sgRNA representation can improve hit detection and enable reliable identification of hits at lower experimental coverage.

5. How does Vivlion design controls for a CRISPR screen?

Vivlion’s scientific team selects and designs appropriate controls based on the CRISPR modality, biological system, readout, and overall experimental design. Controls may include non-targeting sgRNAs, safe-harbor controls, and positive controls with known phenotypes. Following discussion with the customer, additional controls specific to the research objectives can also be incorporated. This tailored approach ensures that the control strategy is aligned with the scientific objectives and requirements of each screen.

6. How is CRISPR screen quality assessed?

Vivlion’s bioinformatics workflow assesses CRISPR screen quality across multiple parameters, including sgRNA abundance and read counts, control- sgRNA enrichment or depletion, replicate correlation, and overall enrichment/depletion patterns. These quality metrics help evaluate library representation, experimental consistency, and the reliability of screening results.

1. What cellular models can be screened by Vivlion?

Vivlion supports CRISPR screening across a broad range of cell models, including both adherent and suspension cell lines. Vivlion also offers screening in three-dimensional (3D) cellular models, including organoids and spheroids.

2. Does Vivlion offer licensed, ready-to-use cell lines for CRISPR screening?

Vivlion has a growing repository of engineered cell lines available for CRISPR screening and is actively expanding this collection. The availability of specific cell lines can be discussed with the Vivlion scientific team based on the screening requirements.

3. Can I provide my own cell line for CRISPR screening?

Yes, Vivlion welcomes customer-provided cell lines and can engineer them for CRISPR screening. Cas nuclease-engineered cell lines provided by the customer can also be used for CRISPR screening. If needed, Vivlion can also source and license a suitable cell line and subsequently perform the required cell engineering to meet the specific screening requirements.