Adeno-Associated Vectors: Overview

Adeno-associated vectors (AAV) are small-size viruses that transduce dividing and non-dividing cells of humans and other species. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. They are very small (approximately 25 nm in diameter) replication-incompetent, non-enveloped viruses. The AAV carries a linear single-stranded (ss)DNA genome of approximately 4.7 kilobases (kb). Several features make AAV an attractive system for delivering a genetic cargo to a cell and tissue-of-interest. First, the vector can transduce both dividing and quiescent cells and persists in an episomal state without integrating into the genome of the host cell. In the wild type virus, however, integration of the vector genome into the host chromosome is common. Over the last 25 years, a significant effort has been devoted to transforming rAAV into one of the platform-of-choice for preclinical and clinical research. Several key milestones have been achieved towards this goal. First, it was demonstrated that the stem-loop-assembled ITRs (see below) are the only cis-acting elements required for viral replication and packaging. This understanding led to the establishment of a packaging cassette which supplies the rep and cap cDNA in-trans. The rep-cap sequence has been replaced with a transgene-of-interest, bringing rAAV packaging capacity close to the 4.7kbs. Moreover, the removal of rep-cap genes from the expression plasmid is important to prevent the re-constitution of wild type AAV genome during production phase. Furthermore, the transfer of the Rep-encoding sequences to the packaging plasmid causes AAV to evade its inherent integration competence. Instead, AAV appears to integrate randomly at a low frequency (≈1%), with the vast majority of its genomes being maintained in the extrachromosomal state. Furthermore, the helper-associated function required for viral replication and production was initially supplied by infecting the producer cells with adenovirus (AdV) or helper simplex virus type 1 (HSV-1). Notwithstanding high efficiency, this approach results in the contamination of the viral preps with adenoviral particles. To circumvent this caveat, researchers engineered a separate plasmid carrying only the necessary helper genes: E1a, E1b, E2a, E4orf6, and viral-associated RNAs. Notably, the HEK293T cells, which are most commonly used for AAV preparations, already have E1a and E1b genes; therefore, these sequences are not included with the helper plasmid. The optimized AAV preparation schedule thus uses three plasmids, namely: vector/expression plasmid carrying a transgene/s-of-interest; rep-cap plasmid and helper plasmid. These plasmids usually are introduced into the producer cells (HEK293/T) by transient transfection method to produce viable AAV preparations. This significant improvement over the older production methods has enabled large-scale preclinical and clinical-grade manufacturing of the recombinant vector for a variety of applications.

Our laboratory utilized a double round-CsCl2 gradient protocol for AAV purification- the only protocol that enables the physical separation of full particles (AAV containing a genome) from empty particles based on their differences in density. Another advantage of this protocol is that its use provides a possibility to purify all serotypes. We use real-time PCR method or fluorescent microscopy to assess the presence of viral genomes in AAV-fractions.
 

We offer adeno-associated vectors in the following formats

• Small AAV preps for in-vitro use (volume-100uL, at the concentration ≥ 5×1011 vg/mL

Great for the transduction into transformed cell lines from human and rodent-origins. The vectors of this grade will be suitable only for in-vitro studies (not suitable for the transduction into neuron cells). Vectors will be supplied in the aliquots of 25 uL. Vectors will be tittered by real time PCR or by counting reporter-expressed cells. 

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 7 days.

• Large AAV preps for in-vitro use (volume-1 mL, at the concentration ≥ 5×1011 vg/mL)

Great for the transduction requiring many samples. Will efficiently transduce transformed cell lines from human and rodent-origins. The vectors of this grade will be suitable only for in-vitro studies (not suitable for transduction into neuron cells). Vectors will be supplied in the aliquots of 200 uL. Vectors will be tittered by real-time PCR or by counting reporter-expressed cells.

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 7 days.

• Small AAV Preps for in-vivo use (volume-100uL, at the concentration ≥ 5 x1012 vg/mL)

This grade is suitable for transduction into delicate cells, such as neurons and for in-vivo studies. The vector grade will greatly fit small in-vivo pilot experiments. Vectors will be supplied in the aliquots of 25 uL. The vectors will be tittered by real-time PCR, or by counting reporter-expressing cells.

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 14 days.

• Mid (Standard) AAV Preps for in-vivo use (volume-500 uL, at the concentration ≥ 5 x1012 vg/mL).

This grade is suitable for the transduction into delicate cells, such as neurons and for in-vivo studies. The vector grade will greatly fit the mid to large in-vivo experiments. The vectors will be supplied in aliquots of 100 uL. The vectors will be tittered by real-time PCR, or by counting reporter-expressing cells.

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 14 days.

• Large AAV Preps for in-vivo use (volume-2 mL, at the concentration ≥ 1 x1012 vg/mL).
  This grade is suitable for the transduction into delicate cells, such as neurons and for in-vivo studies. The vector grade will greatly fit the large in-vivo experiments, including non-human primates (NHPs). The vectors will be supplied in aliquots of 200 uL. The vectors will be tittered by real-time PCR, or by counting reporter-expressing cells.

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 21 days.

• Mega AAV Preps for in-vivo use (volume-10 mL, at the concentration ≥ 1 x1012 vg/mL).
  This grade is suitable for the transduction into delicate cells, such as neurons and for in-vivo studies. The vector grade will greatly fit the large in-vivo experiments in non-human primates (NHPs). The vectors will be supplied in aliquots of 500 uL. The vectors will be tittered by real-time PCR, or by counting reporter-expressing cells.

The QC will also include 

- endotoxin testing

- sterility or bioburden testing

- SDS-PAGE / purity

Production time: 4-6 weeks.

Please note: we cannot guarantee titers if the expression plasmid carrying a transgene-of-interest exceeds 4.8kbs (including the ITRs). The titers of the viral preps will be guaranteed otherwise.
 

Material Safety Data Sheet-AAV.pdf

• Small AAV preps for in-vitro use (volume-100uL, at the concentration ≥ 5×10¹¹ vg/mL)
  Cost: $300

              Production time: 7 days.

• Large AAV preps for in-vitro use (volume-1 mL, at the concentration ≥ 5×10¹¹ vg/mL)
  Cost: $700

              Production time: 7 days.

• Small AAV Preps for in-vivo use (volume-100uL, at the concentration ≥ 5 x10¹² vg/mL)
  Cost: $550

              Production time: 14 days.

• Mid (Standard) AAV Preps for in-vivo use (volume-500 uL, at the concentration ≥ 5 x10¹² vg/mL).                                               
  Cost: $1800

              Production time: 14 days.

• Large AAV Preps for in-vivo use (volume-2 mL, at the concentration ≥ 1 x10¹² vg/mL). 
  Cost: $5000

              Production time: 21 days.
 

• Mega AAV Preps for in-vivo use (volume-10 mL, at the concentration ≥ 1 x10¹² vg/mL).
  Cost: $12,000

              Production time: 4-6 weeks. 

Retroviruses are among the most intensely studied vectors utilized for virus-mediated gene transfer. These studies established the foundation of exploiting retroviral vectors as vehicles for efficient gene delivery into a broad range of tissues and organs. The capacity of efficient integration into the host genome, ability to infect dividing cells and shuttle large genetic payloads, and maintenance of stable, long-term trans-gene expression are attributes that have brought retroviral vectors to the forefront of gene delivery and gene therapy. However, inability of transducing nondividing cells has restricted the employment of retroviruses primarily for gene transfer into hematopoietic cells. As a hallmark, all members of retro/lentiviral vector family are capable of converting single-stranded RNA (ssRNA) of the retrovirus into dscDNA (dsDNA), which can be then stably integrated into the host genome and replicated along with it. As highly evolved parasites retroviruses act in concert with cellular host factors to ensure delivery of their genetic payload into the nucleus, where they exploit host machineries to fulfill replication and long-term expression.

The most common retroviral vectors utilized in our vector core facility are those based on gamma-retrovirus (prototype MLV, or MuLV (murine leukemia viruses)). These retroviral vectors are extremely efficient means to deliver an cDNA-of-interest to a wide range of mammalian cell types. They are by far the easiest and fastest means to deliver genes stably to mammalian cells. The additional advantage of retroviral vectors is that they can be easily adopted for the applications to deliver large libraries of genes, peptides, and RNAi tools.
Retroviral vectors can be generated using transient protocol of transfection (very similar to that used for lentiviral vector production), or via transfection into stable cell lines: Phoenix- MLV cells. (Both methods are commonly used at the facility).

Phoenix helper-free retrovirus producer lines:
Phoenix is a second-generation retrovirus producer line for the generation of helper-free ecotropic and amphotropic retroviruses. The cells have been created in the laboratory of Garry Nolan, Ph.D., (Stanford University) and is based on the HEK293T cells. Phoenix cells were created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic and amphotropic viruses. The lines offered advantages over previous stable systems in that virus can be produced in just a few days.

Figure 1. Production of retroviral vector particles by transfection into Phoenix cells.

Pantropic Retroviral Expression System (Phoenix-GP): The Pantropic- Phoenix cell line is features the HK293T cells that stably-express gamma-retroviral gag and pol proteins. Upon transient transfection of pVSV-G the cell line expresses an envelope glycoprotein from the vesicular stomatitis virus that is not dependent on a cell surface receptor; instead, it mediates viral entry through lipid binding and plasma membrane fusion.
Phoenix cell lines (2nd generation) have several other improvements included the facility to monitor gag-pol production on a cell-by cell basis by introducing an IRES-CD8 surface marker downstream of the reading frame of the gag-pol construct. Thus, CD8 expression is a direct reflection of intracellular gag-pol and the stability of the producer cell population’s ability to produce gag-pol can be readily monitored by flow cytometry. Both Phoenix-Eco and Phoenix-Ampho have been extensively tested for helper virus production and established as being helper-virus free.
The IPRD Viral Vector and Gene Editing Core also offers retroviral vector-packaging using Platinum Packaging Cell Lines to create amphotropic, ecotropic, or pantropic retrovirus (Cell Biolabs):
Plat-A (amphotropic) or Plat-E (ecotropic), which stably expresses both MMLV Gag-Pol and an amphotropic or ecotropic envelope protein, with a retroviral expression vector. These cells require transfection of only an expression vector to produce retrovirus. Pantropic retrovirus can be generated using Plat-GP Retroviral packaging cell line, which stably expresses MMLV Gag-Pol. This cell line can be supplemented with VSV-G pseudotype to generate retroviral vector that is capable of transducing broad-spectrum of cells.
Retroviral vectors can be also generated via transient-transfection protocols; such as calcium-phosphate or polyethylenimine (PEI)-based protocols (similar with production of lentiviral vectors). We use the naïve HEK293T cells to generate retroviral vectors transiently. The Vector Core has amphotropic, ecotropic and pantropic (VSV-G) envelops to generate the respective vectors. We have a collection of gag-pol packaging cassettes to package MLV, MSCV (Murine Stem Cell Virus), and other vector systems.

We offer retroviral vector particles in the following formats
 

• Small Non-Concentrated Retrovirus Preps (volume-10mL, at the concentration 5 x106 vg/mL)

Production time: 7 days
 

• Large Non-Concentrated Retrovirus Preps (volume-100mL, at the concentration ≥ 5 x106 vg/mL). Vectors will be supplied in the aliquots of 10 mL
   

Production time: 7 days.
 

• Concentrated Retrovirus In-Vitro Grade Preps (volume-100uL, at the concentration ≥ 1 x109 vg/mL).

Note: this grade is not suitable for in-vivo use. Vectors will be supplied in the aliquots of 20 uL. Production time: 2 weeks.
 

• Small Concentrated Retrovirus In-Vivo Grade Preps (volume-25uL, at the concentration ≥ 1 x109 vg/mL).
   
• This vector grade greatly fits small in-vivo pilot experiments. Vectors will be supplied in the aliquots of 5 uL.

Note: The cost includes testing for appearance of Replication Competent Retrovirus (RCR) – following FDA recommendations in “Guidance for Industry—Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” (Gunter et al. 1993) and the Vaccines and Related Biological Products Advisory Committee (October 25, 1993). Production time: 2 weeks.
 

• Medium (Standard) Concentrated Retrovirus In-Vivo Grade Preps (volume-150uL, at the concentration ≥ 1 x109 vg/mL).

The cost includes testing for appearance of Replication Competent Retrovirus (RCR)

Production time: 2 weeks.
 

• Large Concentrated Retrovirus In-Vivo Grade Preps (volume-450uL, at the concentration ≥ 2.5 x109 vg/mL).
   

Vectors of this grade will be suitable for large-scale in vivo studies. Vectors will be supplied in the aliquots of 25 uL.
 

The cost includes testing for appearance of Replication Competent Retrovirus (RCR)
 

Production time: 3 weeks
 

GRADES TABLE

Retroviral Vector Production

The IPRD Viral Vector and Gene Editing Core offers high-quality retroviral vectors suitable for a variety of research applications. Our retroviral vectors are designed for stable integration into dividing cells and are produced under rigorous quality control procedures.

Titering and Aliquoting

All retroviral preparations are titered using:

• Flow cytometry or colony-forming assays when reporter or selection markers are present
• Real-time PCR (qPCR) on transduced cells when such markers are absent

Aliquots are provided in 1 mL, 10 mL, or 25 µL volumes depending on prep type. All vectors undergo rigorous quality control to ensure consistency and safety.

Available Prep Formats

1. Non-Concentrated Retroviral Preps (In Vitro Use Only)

Small-Scale Prep

• Volume: 10 mL
• Titer: ≥ 1 × 10⁷ vg/mL
• Aliquot size: 1 mL
• Production time: 7 days
• Applications: Routine transduction of dividing cell lines in vitro
• QC includes:
  • Endotoxin testing
  • Sterility or bioburden testing
  • SDS-PAGE / purity
  • Recombinant-competent retrovirus (RCR) testing

Large-Scale Prep

• Volume: 100 mL
• Titer: ≥ 1 × 10⁷ vg/mL
• Aliquot size: 10 mL
• Production time: 7 days
• Applications: Large-scale in vitro studies
• QC includes: Same as above

2. Concentrated Retroviral Preps (In Vitro Use Only)

In Vitro Grade (Concentrated)

• Volume: 200 µL
• Titer: ≥ 2.5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 14 days
• Applications: High-efficiency in vitro transduction; not suitable for in vivo use
• QC includes:
  • Endotoxin testing
  • Sterility or bioburden testing
  • SDS-PAGE / purity
  • RCR testing

3. Concentrated Retroviral Preps (In Vivo Grade)

These vectors are suitable for in vivo studies in dividing cells. Cost includes testing for replication-competent retrovirus (RCR) following FDA guidance: “Guidance for Industry—Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” (Gunter et al., 1993), and the Vaccines and Related Biological Products Advisory Committee (October 25, 1993).

Small-Scale Prep

• Volume: 50 µL
• Titer: ≥ 2.5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 14 days
• Applications: Pilot in vivo experiments
• QC includes: Full panel including RCR

Medium-Scale (Standard) Prep

• Volume: 200 µL
• Titer: ≥ 2.5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 21 days
• Applications: Dose-response or mid-scale in vivo studies
• QC includes: Full panel including RCR

Large-Scale Prep

• Volume: 1 mL
• Titer: ≥ 5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 28 days
• Applications: Large-scale in vivo studies
• QC includes: Full panel including RCR

Retroviral Vector Prep Summary

About Lentiviral Vectors

Lentiviral vectors (LVs) are HIV-1–based gene delivery systems capable of stably introducing a gene of interest into both dividing and non-dividing cells, with high efficiency in vitro and in vivo.

The LV genome consists of a ~9 kb single-stranded positive-sense RNA molecule, packaged as a homodimer within a protein-enveloped viral particle. Upon binding and entry into the host cell, the viral RNA is reverse transcribed into double-stranded DNA in the cytoplasm. This DNA is then transported into the nucleus, where it integrates into the host genome.

Because lentiviral vectors enable stable integration, the delivered gene is maintained across cell divisions—making them ideal for long-term expression studies and therapeutic applications.

A key advantage of LVs over other viral systems is their ability to transduce non-dividing cells. This sets them apart from:

• Adenoviral and adeno-associated viral (AAV) vectors, which typically do not integrate into the host genome
• Simple retroviral vectors, which require cell division for successful integration

This unique property makes LVs particularly valuable for applications involving terminally differentiated or slowly dividing cell types, including neurons and stem cells.

Lentiviral Vector Production Overview

Lentiviral particles are produced using transient transfection of human embryonic kidney (HEK) 293T cells. These cells express the SV40 Large T antigen, which enhances transgene expression and supports high-titer virus production. Transfection is typically performed using calcium phosphate or polyethylenimine (PEI) protocols.

Three key plasmids (cassettes) are required for lentiviral vector generation:

1. Packaging Cassette

This cassette provides the structural and enzymatic proteins (Gag and Pol) essential for viral particle formation and reverse transcription. It also includes the Rev and Tat proteins, which are critical for RNA processing and expression.

• Rev is required for exporting unspliced or partially spliced viral RNA from the nucleus to the cytoplasm.
• Tat is necessary only if the vector uses the native 5' LTR promoter. If synthetic promoters such as CMV or RSV are used, Tat is not required.
  This second-generation packaging system excludes all HIV-1 accessory genes, enhancing safety and reducing the risk of replication-competent virus formation.

2. Envelope Cassette

This cassette provides the envelope protein, typically the vesicular stomatitis virus glycoprotein (VSV-G), which pseudotypes the vector and enables broad tropism and stability. The envelope is delivered in trans to ensure that it is not incorporated into the viral genome.

3. Expression Cassette

This plasmid carries the gene of interest (e.g., therapeutic or reporter gene), flanked by essential cis-acting elements:

• Long terminal repeats (LTRs): Required for integration into the host genome.
• Psi (Ψ) packaging signal: Ensures selective encapsidation of the vector RNA into viral particles.
• PBS (primer binding site) and PPT (polypurine tract): Cis-elements that support reverse transcription and synthesis of double-stranded DNA.

To enhance safety, the 3' LTR contains a deletion that is transferred to the 5' LTR during reverse transcription, rendering the integrated provirus self-inactivating (SIN). This design ensures that the delivered vector cannot replicate or spread following transduction.

Recommended Further Reading: 

Brown et al. 2020 An Improved Protocol for the Production of Lentiviral Vectors

https://pubmed.ncbi.nlm.nih.gov/33377046/
 

Tagliafierro et al. 2019 Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

https://pubmed.ncbi.nlm.nih.gov/30985756/
 

Vijayraghavan et al 2017 A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

https://pubmed.ncbi.nlm.nih.gov/29286484/

Kantor et al 2014 Methods for Gene Transfer to the CNS

Kantor et al 2014 Clinical Applications involving CNS gene transfer

Schambach et al 2013. Biosafety features of LVs

We offer lentiviral vector particles in the following formats

• Small Non-Concentrated Lentivirus Preps (volume-10mL, at the concentration 2.5 x107 vg/mL)

Great for the transduction into human cell lines, such as HEK293, HELA, HT-29 and others. Vectors will be supplied in the aliquots of 1 mL. Vectors will be tittered by ELISA P24 assay and/or real-time PCR.

Production time: 7 days

• Large Non-Concentrated Lentivirus Preps (volume-100mL, at the concentration ≥ 2.5 x107 vg/mL). Vectors will be supplied in the aliquots of 10 mL. Vectors will be tittered by ELISA P24 assay and/or real-time PCR.

Production time: 7 days.

• Concentrated Lentivirus In-Vitro Grade Preps (volume-100uL, at the concentration ≥ 2.5 x109 vg/mL).
   
• Vectors of this grade will be suitable for in vitro studies and can be used to transduce human non-permissive cells (for example, some primary cells) or cells of not human origin (NIH3T3 fibroblasts, mouse MEFs, mouse C2C12 cells, and others).

Note: this grade is not suitable for the transduction into delicate cells, such as primary neurons or for in-vivo applications. Vectors will be supplied in the aliquots of 20 uL. Vectors will be tittered by ELISA P24 assay and/or real-time PCR. Production time: 2 weeks.

• Small Concentrated Lentivirus In-Vivo Grade Preps (volume-25uL, at the concentration ≥ 2.5 x109 vg/mL).
   
• Vectors of this grade will be suitable for the transduction is suitable for in vitro studies and can be used to transduce human non-permissive cells (for example, some primary cells) or cells of not human origin (NIH3T3 fibroblasts, mouse MEFs, mouse C2C12 cells, and others). This grade is suitable for the transduction into delicate cells, such as neurons and for in vivo studies. This vector grade greatly fits small in-vivo pilot experiments. Vectors will be supplied in the aliquots of 5 uL. Vectors will be tittered by ELISA P24 assay and/or real-time PCR.

Note: The cost includes testing for appearance of Replication Competent Retrovirus (RCR) – following FDA recommendations in “Guidance for Industry—Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” (Gunter et al. 1993) and the Vaccines and Related Biological Products Advisory Committee (October 25, 1993). Production time: 2 weeks.

• Medium (Standard) Concentrated Lentivirus In-Vivo Grade Preps (volume-150uL, at the concentration ≥ 2.5 x109 vg/mL).

Vectors of this grade will be suitable for in-vivo studies and also can be used to transduce human non-permissive cells (for example, some primary cells) or cells of not human origin (NIH3T3 fibroblasts, mouse MEFs, mouse C2C12 cells, and others). Vectors will be supplied in the aliquots of 15 uL. Vectors will be tittered by ELISA P24 assay and real-time PCR.

The cost includes testing for appearance of Replication Competent Retrovirus (RCR)

Production time: 2 weeks.

• Large Concentrated Lentivirus In-Vivo Grade Preps (volume-450uL, at the concentration ≥ 5 x109 vg/mL).

Vectors of this grade will be suitable for large-scale in vivo studies. Vectors will be supplied in the aliquots of 25 uL. Vectors will be tittered by ELISA P24 assay and real-time PCR.

The cost includes testing for appearance of Replication Competent Retrovirus (RCR)

Production time: 3 weeks.

• To generate customized vector at this grade, we will need at least 1.65 mg of plasmid at concentration ≥500 ng/uL prepared using commercially-available kits or using cesium gradient protocol.

GRADES TABLE

VVGEC cannot guarantee titers if not-VSV-G pseudotype is desired.

Material Safety Data Sheet-Lentivirus.pdf

The recommendation of National Institute of Health (NIH) to test concentrated-lentiviral vectors on the subject of appearance of replication-competent retroviruses (RCR) is attached in the document below. The service provided by VVGEC is mandatory and is highlighted here.

Replication-Competent Retrovirus (RCR) assay

The IPRD Viral Vector and Gene Editing Core offers high-quality lentiviral vectors in multiple grades to support a range of in vitro and in vivo applications. All vector preps are quality-controlled and can be customized upon request.

Titering and Aliquoting

All viral preps are rigorously titered for accuracy and reliability:

• Flow cytometry or antibiotic-resistant colony counts are used for vectors expressing appropriate markers.
• For marker-free vectors, real-time PCR (qPCR) on transduced cells is used.
• Vectors are aliquoted in 1 mL, 10 mL, or 25 µL volumes depending on prep type.

Available Prep Formats

1. Non-Concentrated Lentiviral Preps (In Vitro Use Only)

Small-Scale Prep

• Volume: 10 mL
• Titer: ≥ 2.5 × 10⁷ vg/mL
• Aliquot size: 1 mL
• Production time: 7 days
• Applications: Suitable for routine in vitro transduction of transformed cell lines
• QC includes:
  • Endotoxin testing
  • Sterility or bioburden testing
  • SDS-PAGE / purity
  • Recombinant-competent retrovirus (RCR) testing

Large-Scale Prep

• Volume: 100 mL
• Titer: ≥ 2.5 × 10⁷ vg/mL
• Aliquot size: 10 mL
• Production time: 7 days
• Applications: Ideal for large-scale in vitro studies
• QC includes: Same as above

2. Concentrated Lentiviral Preps (In Vitro Use Only)

In Vitro Grade (Concentrated)

• Volume: 200 µL
• Titer: ≥ 5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 14 days
• Applications: High-efficiency in vitro transduction; not suitable for in vivo use
• QC includes:
  • Endotoxin testing
  • Sterility or bioburden testing
  • SDS-PAGE / purity
  • RCR testing

3. Concentrated Lentiviral Preps (In Vivo Grade)

These high-titer preps are suitable for animal studies. Cost includes testing for replication-competent retrovirus (RCR), in accordance with FDA guidelines: "Guidance for Industry—Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors" (Gunter et al., 1993), and the Vaccines and Related Biological Products Advisory Committee (October 25, 1993).

Small-Scale Prep

• Volume: 50 µL
• Titer: ≥ 5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 14 days
• Applications: Small pilot in vivo experiments
• QC includes: Full panel including RCR

Medium-Scale (Standard) Prep

• Volume: 200 µL
• Titer: ≥ 5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 21 days
• Applications: Larger pilot or dose-response studies
• QC includes: Full panel including RCR

Large-Scale Prep

• Volume: 1 mL
• Titer: ≥ 5 × 10⁹ vg/mL
• Aliquot size: 25 µL
• Production time: 28 days
• Applications: Full-scale in vivo studies
• QC includes: Full panel including RCR

Lentiviral Vector Prep Summary

The clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR/Cas) systems has been developed as a powerful molecular technology for plentiful areas of biological research 1. The systems are used by various bacteria and archaea to defend against viruses and other foreign nucleic acids and characterized by tremendous diversity in cas genes, which evolved as a response to selective pressure from their viral invaders and other pathogens 2. This diversity created a broad range of Cas effector proteins, which are significantly varied structurally and functionally. The most interest so far was attracted to Class 2 of the proteins containing a signature Cas9 effector protein, that is in contrast to Class 1 effectors represented by multi-subunit complexes is a single-polypeptide 2, 3, 4, 5. This supported the adaptation of Cas9 which can locate, bind, and cleave double-stranded DNA (dsDNA) targets complementary to its guide crRNA for a wide-range of gene editing applications in mammals shown an incredible impact on many disciplines of human genetics (figure below).

CRISPR/Cas9 composition

CRISPR consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9) or other orthologue (see below). The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified 2, 3, 4, 5. Thus, one can change the genomic target of Cas9 by simply changing the targeting sequence present in the gRNA. CRISPR/Cas9 system can be employed for gene-knockouts, gene activation, repression, epigenetic gene editing (methylation, demethylation) and single-base nucleotide changes (see figure above) 5, 6, 7, 8, 1, 9. Furthermore, the ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies and has been recently utilized for genome-wide screens 10, 11.

The target specificity between the gRNA and the complementary locus requires a presence of Protospacer Adjacent Motif (PAM) that sited downstream and upstream from the target (for Streptococcus pyogenes Cas9 and Cpf1, respectively) 2, 3, 4, 5, 12. The PAM sequence is necessary for target binding and the exact sequence is dependent upon the species of Cas9 (5′ NGG 3′ for Streptococcus pyogenes Cas9 and NNGRRT for Cpf1) 2, 5, 12. Once expressed, the Cas9 protein and the gRNA form a riboprotein complex. The Cas9-gRNA complex will bind any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cleave DNA. The zipper-like mechanisms of spacer-photospacer binding has been proposed shown the absolute requirement of 10-PAM 5’-adjoined nucleotides called a “seed” sequence 4, 5. Any change in the seed sequences may negatively affect the affinity of DNA-spacer interactions. Furthermore, Cas9 will only cleave the target if sufficient homology exists between the gRNA spacer and target sequences. The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH, which cut the opposite strands of the target DNA 2, 5, 3, 4.

DNA repair pathways activated by CRISPR/Cas9- mediated DSBs

The cleavage will result in a double strand breaks (DSBs) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence) 2, 5, 4. Two DNA repair machineries: error-prone non-homologous end joining (NHEJ) and less efficient, but highly accurate homology directed repair (HDR) are undergo activation following DNA break. The NHEJ repair pathway is the most active repair mechanism, capable of rapidly repairing DSBs. The pathway frequently results in small nucleotide insertions or deletions (InDels) at the DSB site and in most cases, NHEJ gives rise to small InDels in the target DNA which result in in-frame amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene.

By contrast, the DSBs can be repaired by HDR- pathway. To insert or replace a DNA sequence near the break site, a DNA fragment to be used as a template for repair is introduced (reviewed in 1. The repair template contains homology to the regions flanking the DSB- repair of the break leads to insertion of the repair template without introducing extraneous bases. Thus via HDR, scarless insertion of DNA can be introduced to create precise deletions, base substitutions, or insertion of coding sequences for epitope tags, such as fluorescent proteins. This technology has also been applied to engineering genomes of other eukaryotes, such as yeast, flies and zebrafish. The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. To enhance HDR, enabling the insertion of precise genetic modifications, several approaches were utilized 13, 14. The researchers demonstrated that suppression of the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the co-expression of adenovirus 4 E1B55K and E4orf6 proteins might improve the efficiency of HDR 5 to10- fold 14. The low efficiency of HDR has several important practical implications. First, since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally, and if necessary, isolate clones containing the desired genotype. The two pathways of DNA repair highlighted in the figure that below:

CRISPR/Cas9 is highly specific when gRNAs are designed correctly, but specificity is still a major concern, particularly as CRISPR is being developed for clinical use. The specificity of the CRISPR system is determined in large part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and need to be considered when designing a gRNA for your experiment ³, ⁴.

In addition to optimizing gRNA design, specificity of the CRISPR system can also be increased through modifications to Cas9 itself. First, Cas9 generates DSBs through the combined activity of two nuclease domains, RuvC and HNH. The exact amino acid residues within each nuclease domain that are critical for endonuclease activity are known (D10A for RuvC and H840A for HNH in S. pyogenes Cas9) and modified versions of the Cas9 enzyme containing only one active catalytic domain (called “Cas9 nickase”) have been generated 3, 4. Cas9 nickases still bind DNA based on gRNA specificity, but nickases are only capable of cutting one of the DNA strands, resulting in a “nick”, or single strand break, instead of a DSB. DNA nicks are rapidly repaired by HDR using the intact complementary DNA strand as the template. Thus, two nickases targeting opposite strands are required to generate a DSB within the target DNA (often referred to as a “double nick” or “dual nickase” CRISPR system) 3, 4, 15. This requirement dramatically increases target specificity. Second, more recently discovered Cpf1 endonuclease demonstrated to be more specific than the SpCas9 enzyme 16, 12. Furthermore, efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of SpCas9-HF1 and eSpCas9 (1.0 and 1.1) variants that contain amino acid substitutions predicted to weaken the energetics of target site recognition and cleavages 17, 18. Using single-molecule Förster resonance energy transfer (smFRET) experiments, Doudna’s group most recently shown that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets 19. They found that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, the researchers designed a new hyper-accurate Cas9 variant (HypaCas9) that has demonstrated high genome-wide specificity without compromising on-target activity in human cells 19.

Activation or Repression of Target Genes Using CRISPR/Cas9

In addition to utility of CRISPR/Cas9 for direct manipulation within DNA sequences, investigators modified the system to alter the regulation of a target gene. One approach to increase expression of a specific gene is to tether the dead- version of Cas9 (harbored D10A and H840A mutations) dCas9:sgRNA complex to a transcriptional activator and program it to bind near the transcriptional start site of a gene of interest 7, 9, 6, 20, 21. For example, the transcriptional activation domain VP64, which consists of four tandem copies of the Herpes Simplex Viral Protein 16 (VP16), attached to the C terminus of dCas9 can be used to increase the expression of a wide variety of different genes. Similarly, KRAB repressor can be fused to the dCas9 creating a robust platform for gene-specific transcriptional silencing. Recently, more sophisticated approaches has been developed for highly efficient regulation of gene-expression programs. These include- coexpression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, dCas9 fused to several different activation domains in series (e.g. dCas9-VPR) or co-expression of dCas9-VP64 with a “modified scaffold” gRNA and additional RNA-binding “helper activators” (e.g. SAM activators) 22, 23, 9, 6. Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.

Activation and repression of a target gene: RNA-programmed gene activators can be assembled through the direct fusion of dCas9 with the transcriptional activators VP64, or indirect- via MS2 binding motif- with transcriptional activators p65-HSF, NF-kb and others (not shown here- the histone acetyltransferase enzyme p300 or the DNA demethylase Tet1) ⁷, ⁹, ⁶, ²⁰, ²¹. Alternately, transcriptional repressors such as KRAB, de-novo methyltransferase 3A and the histone demethylase LSD1 can be fused either directly or indirectly to repress a target gene ²⁰, ²⁴, ²⁵.

Delivery platforms

Many viral and non-viral methods for delivery of CRISPR/Cas9 gene-editing tools has been recently developed (reviewed in ²⁶, ²⁷, ²⁸). The obvious advantage of viral-mediated gene-transfer is that it deliver a therapeutic cargo in highly efficient manner, currently unachievable by non-viral methods and strategies. Furthermore, initially safety concerns including high immunogenicity and an increased risk of insertional mutagenesis have been recently addressed with the development of more advance and safe viral tools ²⁹, ³⁰.

Non-viral vectors are generally lower in efficiency but have the advantage of diverse available chemistry, capacity for functionalization and targeting, and ease of manufacturing. Both delivery systems have seen success in specific applications. The main advantage of non-viral mediated gene-delivery stems from its transient capability. Indeed, transient platforms including DNA plasmids, messenger RNA (mRNA), and ribonucleoprotein (RNP)-molecules utilized for CRISPR/Cas9 delivery have been shown to be highly advantageous for minimizing non-specific effects of the CRISPR/Cas9 system (reviewed in ²⁶, ²⁷). Indeed, the high turnover rate of rapidly degraded transiently delivered CRISPR/Cas9 corresponded to a reduced rate of off-target mutations. Furthermore, Cas9-sgRNA RNPs-mediated DNA cleavage is followed by their almost instant degradation and clearance from the cells, which suggests that gene editing might only require a short-term presence of its components ³¹.

However, low transduction efficiency remains a serious limitation of RNPs and other non-viral delivery platforms. To overcome this limitation, a lentivirus-pre-packaged Cas9 protein (Cas9P LV) system was developed recently and was shown to be effective for disruption of gene expression in naïve T cells32. Significantly, transiently delivered Cas9 showed high target specificity and induced no measurable InDels at off-target DNA sites. However, production titers of the prepackaged-Cas9 system were observed to be lower than those of conventional LVs32.

Lentiviral vectors (LVs) are an important means of delivering CRISPR/Cas9 components due to their ability to accommodate large DNA payloads and efficiently transduce a wide range of dividing and non-dividing cells. LVs also display low cytotoxicity and immunogenicity and have a minimal impact on the life cycle of the transduced cells (reviewed in 29). Such features have led to LVs being used as the gene-editing regimen of choice to treat HIV-1, HBV and HSV-1 infections, as well as to correct defects underlying human hereditary diseases, such as cystic fibrosis ³³, ³⁴, ³⁵, ³⁶. Despite these successes, this system suffers from significant drawbacks. Permanently expressed CRISPR/Cas9 may facilitate undesirable off-target effects, hindering their utility for genome-editing applications that require high levels of precision. Indeed, rise of promiscuous interactions with off-target genes due to excess gRNA/Cas9 is well-documented ¹⁰, ³¹. Furthermore, sustained expression of gRNA/Cas9 in vitro increases the tolerability of mismatches in the guide-matching region and the protospacer adjacent motif (PAM), thereby promoting non-specific double-strand breaks (DSBs) ³⁷, ³⁸. Along the same lines, the ratio of insertions and deletions (InDels) at off-target vs. target sites in vivo increases with higher Cas9 and gRNA concentrations 3. These observations suggest that non-integrating vectors would be desired for delivery CRISPR/Cas9 components if the enhance safety of gene-editing manipulations are pursued.

Integrase-deficient lentiviral vectors

Integrase-deficient lentiviral vectors (IDLVs) have garnered significant interest among researchers for precise in vivo analysis of genetic diseases, since they significantly reduce the risk of insertional mutagenesis inherent in integrating delivery platforms (reviewed in {Kantor, 2014 #53}, {Kantor, 2011 #26}, {Nelson, 2016 #66}. IDLVs are an ideal platform for delivery of large genetic cargos where only transient expression of the transgene is desired (reviewed in ⁶, ²², ²³, ²⁴), ²⁵, ²⁶, ²⁷, ²⁸, ²⁹, ³⁰, ³¹, ³². IDLVs were successfully employed in the past in mouse models as gene replacement therapies for degenerative retinal disease, hemophilia B (³⁶, ³⁷); they show high efficacy in cancer immunotherapy- setting as a means of inducing protective immune responses to human pathogens ³⁸, ³⁹, ⁴⁰. Furthermore, a growing body of literature describes IDLVs carrying zinc-finger nucleases as an effective means of gene editing for clinical and basic science applications ²⁸, ³⁰, ³¹, ³². For instance, Lombardo and colleagues have successfully employed non-integrating vectors as a means of avoiding genotoxicity associated with continuous expression of zinc-finger nucleases (ZFNs), and for delivering the donor DNA template required for DNA repair-mediated gene editing ²⁸. These researchers demonstrated that the IDLV-ZFNs system is capable of effectively disrupting expression from the gene encoding the HIV-1-coreceptor CCR5. Additionally, Joglekar and colleagues³⁰ successfully employed IDLVs to deliver ZFNs and donor templates for site-specific gene modification at the human adenosine deaminase (hADA) locus in primary T-lymphocytes. Most recently, Hoban and colleagues demonstrated efficient gene editing of the mutated human β-globin gene in CD34+ hematopoietic stem and progenitor cells by co-delivering CRISPR/Cas9 reagents and donor templates via IDLVs41.

We recently developed novel IDLVs for delivering Cas9 and sgRNA through a single vector and demonstrated that the novel vector enables facile and robust gene editing in in-vitro and in-vivo settings – which is particularly advantageous for developing translatable gene therapy products {Ortinski, 2017 #83}. Furthermore, we demonstrate that the IDLV-CRISPR/Cas9 system is expressed transiently and has a significantly lower capacity to induce off-target mutations than its integrating counterparts have {Ortinski, 2017 #83}.

The image copied from Ortinski et al. 2017; published in Molecular Therapy Methods and Clinical Development. (A) A schematic map of the vector cassette. A shorter version of pLentiCRISPRv2 was created to include a unique BsrGI restriction enzyme site flanked by two BsmBI sites to be used for cloning sgRNAs. Other regulatory elements of the vectors include a primer-binding site (PBS), splice donor (SD) and splice acceptor (SA), central polypurine tract (cPPT) and PPT, Rev Response element (RRE), WPRE, and the retroviral vector-packaging element, psi (ψ) signal. A human cytomegalovirus (hCMV) promoter, a core-elongation factor 1α promoter (EFS-NC), and a human U6 promoter are highlighted. (B) Production titers of the vectors with (novel) and without (parental) Sp1 binding sites as determined by p24ELISA assay. The results shown increase in production of novel IDLVs and (C) ICLV vectors (the functional titer has been determined by counting puromycin-resistant colonies).

Adenoviruses and adeno-associated vectors (AAVs)

Adenoviruses and AAVs have also been broadly investigated for their potential use in vitro and ex vivo (reviewed in {Kantor, 2014 #53}, {Kantor, 2011 #26}). The production titers are traditionally high with AAVs which makes this viral platform to be the most used viral vector for genome engineering to date (reviewed in 26). In addition, being transient delivery platform highlights one of the main advantages of AAV vectors. First publications utilized AAV for CRISPR/Cas9 delivery used SpCas9- for example, Platt and colleagues39 successfully packaged the endonuclease and sgRNA into viral particles for in vivo modeling of loss-of-function mutations in P53 and LKB1 genes in mouse lung adenocarcinomas. However, the large size of the SpCas9 gene (4.2kb) imposes a significant burden on the packaging capacity of AAVs. To overcome this bottleneck, Gang Bao’s group recently developed a split-intein Cas9 system that can be separated into two AAV cassettes 40. This approach allows for increased in overall packaging capacity but necessitates production and co-transduction of two AAV vectors. The discovery of a shorter, but equally potent Cas9 enzyme derived from Staphylococcus aureus (SaCas9) (see above) led to development of SaCas9/guide RNA system that could be efficiently packaged and delivered by AAV vectors 41. This system was shown to efficiently target the cholesterol regulatory gene PCSK9 in the mouse liver 41.

The IPRD Viral Vector and Gene Editing Core is pleased to offer a comprehensive package of services involved creation and production of customized AAV, IDLV and LV/RV vectors for delivery CRISPR/Cas9 gene-editing tools. Our CRISPR/Cas9 vectors are suitable for both in-vitro and in-vivo studies. In addition, we guarantee highest- quality standards and safety of the vectors. The core owns comprehensive collection of CRISPR/Cas9 plasmids acquired from Addgene and created at the facility. Our vectors are production/titer optimized.

CRISPR/Cas9 plasmids received from Addgene are not available for the distribution.

References:

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2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 (2012).
    
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5. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826, doi:10.1126/science.1232033 (2013).
    
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8. Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov 2, 16009, doi:10.1038/celldisc.2016.9 (2016).
    
9. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583-588, doi:10.1038/nature14136 (2015).
    
10. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84-87, doi:10.1126/science.1247005 (2014).
     
11. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80-84, doi:10.1126/science.1246981 (2014).
     
12. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759-771, doi:10.1016/j.cell.2015.09.038 (2015).
     
13. Song, J. et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun 7, 10548, doi:10.1038/ncomms10548 (2016).
     
14. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33, 543-548, doi:10.1038/nbt.3198 (2015).
     
15. Tycko, J., Myer, V. E. & Hsu, P. D. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity. Mol Cell 63, 355-370, doi:10.1016/j.molcel.2016.07.004 (2016).
     
16. Fagerlund, R. D., Staals, R. H. & Fineran, P. C. The Cpf1 CRISPR-Cas protein expands genome-editing tools. Genome Biol 16, 251, doi:10.1186/s13059-015-0824-9 (2015).
     
17. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495, doi:10.1038/nature16526 (2016).
     
18. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84-88, doi:10.1126/science.aad5227 (2016).
     
19. Chen, J. S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407-410, doi:10.1038/nature24268 (2017).
     
20. Gilbert, L. A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159, 647-661, doi:10.1016/j.cell.2014.09.029 (2014).
     
21. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183, doi:10.1016/j.cell.2013.02.022 (2013).
     
22. Huang, Y. H. et al. DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A. Genome Biol 18, 176, doi:10.1186/s13059-017-1306-z (2017).
     
23. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12, 326-328, doi:10.1038/nmeth.3312 (2015).
     
24. McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol Open 5, 866-874, doi:10.1242/bio.019067 (2016).
     
25. Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods 12, 401-403, doi:10.1038/nmeth.3325 (2015).
     
26. Nelson, C. E. & Gersbach, C. A. Engineering Delivery Vehicles for Genome Editing. Annu Rev Chem Biomol Eng 7, 637-662, doi:10.1146/annurev-chembioeng-080615-034711 (2016).
     
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31. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24, 1012-1019, doi:10.1101/gr.171322.113 (2014).
     
32. Choi, J. G. et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther 23, 627-633, doi:10.1038/gt.2016.27 (2016).
     
33. Wang, W. et al. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection. PLoS One 9, e115987, doi:10.1371/journal.pone.0115987 (2014).
     
34. Kennedy, E. M. et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology 476, 196-205, doi:10.1016/j.virol.2014.12.001 (2015).
     
35. Roehm, P. C. et al. Inhibition of HSV-1 Replication by Gene Editing Strategy. Sci Rep 6, 23146, doi:10.1038/srep23146 (2016).
     
36. Bellec, J. et al. CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells. Curr Gene Ther 15, 447-459 (2015).
     
37. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31, 839-843, doi:10.1038/nbt.2673 (2013).
     
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40. Truong, D. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res 43, 6450-6458, doi:10.1093/nar/gkv601 (2015).
     
41. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191, doi:10.1038/nature14299 (2015).
     
42. Kennedy, E. M. et al. Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic. Methods 91, 82-86, doi:10.1016/j.ymeth.2015.08.012 (2015).

Note: CRISPR/Cas9 plasmids received from Addgene are not available for the distribution

The core has a comprehensive collection of LVs and AAVs vectors expressed CRISPR-Cas9 components for gene editing- gRNA with or without Cas9 nuclease; WT-Cas9; nickase-Cas9; inducible-Cas9 and dCas9.

The collection derives from in-house developed expression cassettes (backbone-optimized & titer-improved) (please see Ortinski et al, 2017 in Molecular Therapy Methods and Clinical Dev) http://www.cell.com/molecular-therapy-family/methods/abstract/S2329-0501(17)30057-8 or obtained from other investigators via Addgene repository. The vectors contain fluorescent markers for direct visualization of expression and/or antibiotics-resistance genes (puromicin, blastidin and others) which can be used if selection of transduced cells is desired. The vectors support single or paired gRNA-CRISPR approach for efficient gene editing manipulations. Most of the viral platforms are validated - we will be able to guarantee high-level of CRISPR/Cas9 expression.

Lentiviral vectors- backbones
 

1. pBK109- short version derived from pLentiCRISPR/v2 ready-for-gRNA-cloning (BsmBI)
    
2. pBK301- short version of pLentiCRISPR/v2 with 2 Sp1 binding sites upstream of U6p- gRNA cloning site BsmBI
    
3. pBK175- short version of pLentiCRISPR/v2 with 4 Sp1 binding sites upstream of U6p- gRNA cloning site BsmBI
    
4. pBK110- pLentiCRISPR/v2 Luciferase-gRNA
    
5. pBK104- pLentiCRISPR/v2 GABA a2-subunit-gRNA
    
6. pBK83- pLentiCRISPR/v2 HDAC1-1 –gRNA
    
7. pBK84-pLentiCRISPR/v2 HDAC1-2 –gRNA
    
8. pBK85-pLentiCRISPR/v2 HDAC1-3 – gRNA
    
9. pBK86-pLentiCRISPR/v2 GFP1 – gRNA
    
10. pBK87-pLentiCRISPR/v2 GFP2 – gRNA
     
11. pBK88-pLentiCRISPR/v2 GFP3 – gRNA
     
12. pBK189- pLentiCRISPR/v2 with GFP-gRNA
     
13. pBK198- short version of pLentiCRISPR/v2 with 2 Sp1 binding sites with GFP-gRNA
     
14. pBK180- rtTA3 Blasticidin third generation rtTA3 can be used for tet-Cas9 system (next plasmid)
     
15. pBK185- tet- inducible Cas9- all-in-one short- with BsmBI for cloning sgRNA
     
16. pBK195- tet- inducible Cas9- all-in-one short- with GFP-gRNA
     
17. pCW-Cas9 Tet ON Plasmid #50661; Addgene- https://www.addgene.org/50661/
     
18. pBK109easy -GFP pLenti-SpCas9gRNA with GFP all-in-one
     
19. pBK97- SpCas9 nickase- all-in-one lentiviral backbone; titer-optimized backbone; ready for gRNA cloning BsmBI
     
20. pBK456- dCas9- all-in-one lentiviral backbone; titer-optimized backbone; ready for gRNA cloning BsmBI
     
21. pBK109easy -GFP pLenti-SpCas9gRNA with GFP all-in-one
     
22. pBK109BL- short version of pLentiCRISPR/v2 gRNA cloning site BsmBI with blasticidin
     
23. pBK114- pLenti-Cas9-GFP
     
24. pLentiCRISPR/v2 Plasmid #52961; Addgene- https://www.addgene.org/52961/
     
25. pLenti-multi-CRISPR; Plasmid #85402; Addgene- https://www.addgene.org/85402/
     
26. LentiCRISPRv2-mCherry; Plasmid # 99154; Addgene- http://www.addgene.org/99154/
     
27. pLenti-pU6-sgRNA Ef1alpha-Puro-T2A-BFP Plasmid #84832; Addgene- http://www.addgene.org/84832/
     
28. pLenti-sgRNA; Plasmid #71409; Addgene- http://www.addgene.org/71409/
     
29. pLenti-CRISPR.EFS.tRFP; Plasmid #57819; Addgene- http://www.addgene.org/57819/
     
30. lentiCas9-Venus; Plasmid # 70267; Addgene- http://www.addgene.org/70267/
     
31. pL-CRISPR.EFS.GFP Plasmid #57818; Addgene- https://www.addgene.org/57818/
     
32. lentiCas9-Blast Plasmid #52962; Addgene- https://www.addgene.org/52962/
     
33. lentiCas9-Puro Plasmid #52963; Addgene-http://www.addgene.org/52963/
     
34. lentiCas9-EGFP; Plasmid #63592; Addgene- https://www.addgene.org/63592/
     
35. lentiCas9n(D10A)-Blast; Plasmid #63593; Addgene- http://www.addgene.org/63593
     
36. lenti-dCAS-VP64-Blast; Plasmid #61425; Addgene- http://www.addgene.org/61425/
     
37. pHAGE TRE dCas9-VP64; Plasmid #50916; Addgene- http://www.addgene.org/50916/
     
38. lenti-TRE-KRAB-dCas9-IRES-BFP; Plasmid #85449; Addgene- http://www.addgene.org/85449/
     
39. plenti- hUbC-dCas9 VP64-T2A-GFP; Plasmid #53192; Addgene- http://www.addgene.org/53192/
     
40. pKLV-U6gRNA(BbsI)-PGKpuro2ABFP; Plasmid #50946; Addgene- http://www.addgene.org/50946/
     
41. plentiSAMv2; Plasmid #75112; Addgene- http://www.addgene.org/75112/
     
42. pHR-SFFV-KRAB-dCas9-P2A-mCherry; Plasmid #60954; Addgene- http://www.addgene.org/60954/
     
43. pLV-dCas9-KRAB-PGK-Hyg- Plasmid #83890; Addgene- http://www.addgene.org/83890/
     
44. pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP; Plasmid #71237; Addgene- http://www.addgene.org/71237/
     
45. pHAGE EF1α dCas9-KRAB; Plasmid #50919; Addgene- http://www.addgene.org/50919/
     
46. lenti sgRNA-MS2-Zeo; Plasmid #61427; Addgene- http://www.addgene.org/61427/
     
47. lenti sgRNAMS2- puro optimized backbone; Plasmid #73797; Addgene- http://www.addgene.org/73797/
     
48. pHAGE-TO-nmdCas9-3XGFP; Plasmid #64109; Addgene- http://www.addgene.org/64109/
     
49. pLV-dCas9-p300-P2A-Puro; Plasmid #83889; Addgene- http://www.addgene.org/83889/
     
50. Lenti_sgRNA-EFS-GFP; Plasmid #65656; Addgene- http://www.addgene.org/65656/
     
51. Lenti-AsCpf1-Blast; Plasmid #84750; Addgene- http://www.addgene.org/84750/
     
52. LentiCRISPRv2Cre; Plasmid #82415; Addgene- http://www.addgene.org/82415/
     
53. Exp_v-pcDNA3.1-hAsCpf1; Plasmid #69982; Addgene- https://www.addgene.org/69982/
     
54. Exp_v-pSimpleII-U6- gRNA cloning site BsmBI-NLS-NmCas9-HA-NLS plasmid # 47868; Addgene https://www.addgene.org/47868/
     
55. Exp_v-pSaCas9_GFP; Plasmid # 64709; Addgene- https://www.addgene.org/64709/
     
56. Exp_v-Csy4-T2A-Cas9-NLS; Plasmid # 53371; Addgene- https://www.addgene.org/53371/
     
57. Exp_v-ppcDNA3.1-hFnCpf1; Plasmid #69976; Addgene- https://www.addgene.org/69976/
     
58. Exp_v-eSpCas9(1.1)- Plasmid #71814; Addgene-https://www.addgene.org/71814/
     
59. Exp_v-SpCas9-HF1 (high fidelity Cas9); Plasmid #72247; Addgene- https://www.addgene.org/72247/

1. AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA Plasmid # 61591; Addgene- https://www.addgene.org/61591/
    
2. BK- pAAV-EFS-NC- SpCas9-NLS-Poly(A)
    
3. BK- pAAV-CMV-SpCas9- NLS-Poly(A)
    
4. BK- pAAV-hSyn -SpCas9- NLS-Poly(A)
    
5. BK- pAAV- -SpCas9- NLS-Poly(A)
    
6. pAAV- nEF promoter- Cas9; Plasmid #87115; Addgene- http://www.addgene.org/87115/
    
7. pAAV-pMecp2-SpCas9 Plasmid #60957 http://www.addgene.org/60957/
    
8. pX602-AAV-TBG::NLS-SaCas9-NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA Plasmid #61593; Addgene- http://www.addgene.org/61593/
    
9. pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA; Plasmid #61591; Addgene- http://www.addgene.org/61591/
    
10. AAV-NFS-NC: NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
     
11. AAV-Syn: NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
     
12. pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA; Plasmid #61592; Addgene- http://www.addgene.org/61592/
     
13. pJEP12-AAV-H1/TO(dox-regulated)-L-sgRNA(Empty)-CMV-TetR-P2A-eGFP-KASH-pA; Plasmid #82704; Addgene- http://www.addgene.org/82704/
     
14. pX603-AAV-CMV::NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA; Plasmid #61594; Addgene-http://www.addgene.org/61594/
     
15. AAV-NFS-NC:NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
     
16. AAV-Syn:NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
     
17. AAV-MeCp2:NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
     
18. AAV-NFS-NC:NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
     
19. AAV-NFS-NC:NLS-dSaCas9Nickase-NLS-3xHA-bGHpA
     
20. AAV-CMV:NLS-dSaCas9Nickase-NLS-3xHA-bGHpA
     
21. AAV-CAG:NLS-dSaCas9Nickase NLS-3xHA-bGHpA
     
22. AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR; Plasmid #60231; Addgene- http://www.addgene.org/60231/
     
23. AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR; Plasmid #60226; Addgene-http://www.addgene.org/60226/
     
24. AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL); Plasmid #60224; http://www.addgene.org/60224/
     
25. BK- pAAV-TRE – SpCas9-NLS-Poly(A)
     
26. BK- pAAV-TRE- SaCas9-NLS-Poly(A)
     
27. BK- pAAV-TRE – dSpCas9-NLS-Poly(A)
     
28. BK- pAAV-TRE – dSaCas9-NLS-Poly(A)
     
29. BK- pAAV-TRE – SpCas9Nickase-NLS-Poly(A)
     
30. BK- pAAV-TRE – SaCas9Nickase-NLS-Poly(A)

The IPRD Viral Vector and Gene Editing Cores deliver advanced solutions for biomedical research and therapeutic development. Our expertise in CRISPR/Cas technologies, viral vector engineering, and customized cell and animal model generation empowers investigators to accelerate discovery and translational applications.

We offer a comprehensive portfolio of services that includes the design and production of CRISPR/Cas vectors, generation of genetically engineered cell lines, and customized editing strategies using the latest technologies such as base editing, prime editing, and epigenome editing. Our team is here to partner with you—whether you are developing basic research tools, building disease models, or pursuing therapeutic targets.

1. Gene Knockout Cell Line Services

Our core provides fully customizable CRISPR/Cas9-based knockout services in a wide range of mammalian cell lines, including difficult-to-transfect and tumor-derived lines. We deliver functionally validated, long-term stable KO models to researchers at Florida State University and to institutions and companies worldwide.

Our platform includes:

• High-throughput gRNA screening and optimized RNP delivery
• Single- and multi-gene knockout strategies
• Targeted fragment deletions
• End-to-end support from gRNA design to functional validation

Standard workflow:

• Host Cell Characterization
  Includes clonability assays, antibiotic resistance profiling, and optimization of transfection/transduction.
• gRNA Design & KO Vector Construction
  Guides are designed to target conserved exons or critical functional domains of the gene-of-interest.
• Transfection & Transduction
  Using optimized techniques and house-developed lentiviral and adeno-associated vectors.
• Clone Screening
  Selection via antibiotic resistance or FACS; expansion of monoclonal or polyclonal populations.
• Validation
  Characterization via Western blotting, qPCR, ELISA, or reporter assays.

2. Specialized Gene Editing Services

Multiplexed Knockouts
Generate cell lines with multiple gene deletions using pooled or sequential editing strategies.

Disease Modeling
Create precise genetic alterations to model rare or common human diseases in vitro.

Custom CRISPR Libraries
Design and produce small- to large-scale gRNA libraries for functional genomics studies.

Nuclease Activity Validation
Quantify and optimize CRISPR nuclease performance using a range of molecular and cellular assays.

3. Base Editing and Prime Editing

We offer advanced genome editing platforms based on base editors and prime editors—technologies that allow precise, single-nucleotide changes without introducing double-stranded breaks.

Base Editing

Our cytidine and adenine base editors enable targeted C-to-T or A-to-G conversions with minimal off-target effects. These editors are delivered using viral vectors customized for your cell type and application.

• Editing window: 6–10 nucleotides
• Delivery via AAV, lentivirus, or electroporation
• Applications: point mutation correction, functional SNP studies, regulatory region targeting

Prime Editing

We design and deliver pegRNA constructs that enable precise insertions, deletions, or substitutions.

• pegRNA components: spacer, scaffold, reverse transcription template (RTT), primer binding site (PBS)
• nCas9-reverse transcriptase system enables templated DNA repair
• Suitable for applications requiring programmable, high-fidelity edits

With over 15 years of experience in guide RNA design, viral delivery, and construct engineering, we support projects across a wide range of biological systems.

4. Epigenome Editing

Epigenetic modifications enable control of gene expression without altering the underlying DNA sequence. We offer full-service epigenome editing platforms using dCas9-based tools fused to:

• DNA methyltransferases and demethylases
• Histone acetyltransferases and deacetylases

Workflow includes:

1. Target Identification – Defining loci relevant to gene regulation or disease
2. Tool Construction – Building custom dCas9 fusion proteins
3. Vector Development – Cloning into expression vectors for delivery
4. Delivery – Viral, electroporation, or nanoparticle-based methods
5. Selection & Expansion – Isolation of modified clones
6. Validation – ChIP, bisulfite sequencing, and gene expression profiling

5. Vector and Construct Resources

We maintain an extensive collection of >3,000 in-house optimized CRISPR/Cas constructs for gene knockout, base editing, and more. These are:

• Available directly from the core
• Deposited in the Addgene repository (https://www.addgene.org)
• Linked to the NGVB repository (https://ngvbcc.org/ReagentRepository)

Our design and production workflows are supported by 10 patent applications and numerous peer-reviewed publications and funded grants.

Let’s Collaborate
We welcome collaborations with academic, government, and industry partners. Whether your goal is to explore gene function, build disease models, or develop precision therapeutics, our core is ready to support your success.

Contact us today to discuss your project needs and receive a customized consultation.