PIK3CD CRISPR All-in-one AAV vector set (with saCas9)(Mouse)
Unit
3x1.0μg DNA
Description
abm's Knockout sgRNA vectors and viruses are ready to use in your CRISPR Gene Knockout experiment. These vector and virus products come as a set of three sgRNA targets which are designed to guide Cas9 to cleave exonic gDNA resulting in frameshift mutations and ultimately gene knockout. Available constructs include All-in-One (Cas9 and sgRNA expression) and sgRNA only (no Cas9 expression) – navigate to Custom Option to select the latter. Our CRISPR vectors and viruses are also available in a variety of formats including Lentivirus, AAV and Non-Viral.
☛ View our CRISPR Workflow Guide to learn how to use our CRISPR KO products and identify key stages in delivery, selection, and screening.
Caution
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information.
The lentivirus should be aliquoted into smaller working volumes and then stored at -80°C. Lentivirus is sensitive to storage temperature and to freeze/thaws. It can lose up to 5% or more activity with each freeze/thaw. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
abm lentiviral transfer vectors use the third generation lentivirus system. It is based on the inactivated HIV genome. Note that our lentivirus packaging plasmids cannot be integrated into the host and are transiently expressed.
MOI (Multiplicity of Infection) refers to the number of viral particles per cell used in the infection, e.g. an MOI of 5 indicates that there are five infectious units (IU) or transducing units (TU) for every cell. MOI is determined by calculating the numbers of viral particles added per well then divide this number by the number of cells seeded into the well. We also recommend transducing the cells with a range of MOIs as different cell types may require different MOIs for successful transduction.
MOI = Product Titer (IU/ml) x Virus Volume (ml) / Total Cell Number
Optimal cell density for lentiviral transduction is generally 50-70% confluent for most cell types. Certain cell lines may require the optimal cell density to be determined experimentally.
These are medium-high copy plasmids and should be propagated in a cloning E. coli strain such as DH5α. Typical yields from a 250ml culture is 300-500µg plasmid DNA.
Standard delivery of abm’s vectors are in liquid format supplied in TE buffer (10mM Tris, 1mM EDTA, pH 8.0). Our vectors can also be ordered and delivered in agar stabs for an additional fee.
abm's lentiviral vectors are compatible with 2nd and 3rd generation packaging systems. We recommend abm’s Second Generation Packaging Mix (Cat. No. LV003) for users who want to achieve higher titer lentivirus as only 3 plasmids are required for virus production instead of 4 plasmids. We also recommend abm’s Third Generation Packaging Mix (Cat. No. LV053) for users concerned with virus biosafety, as the viral packaging elements are further split into 4 plasmids thus reducing probably of producing wild type virus.
Please note, only packaging mixes produced by abm have been tested in house and therefore carry our guarantee for high titer virus production. If it is desirable to use other packaging plasmids obtained from a different source, the compatibility must be tested and determined by the end user.
Yes, all of abm’s lentiviral vectors contain the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) upstream of the 3’LTR. This element when transcribed creates a tertiary structure which enhances gene expression.
MOI stands for multiplicity of infection. Theoretically, an MOI of 1 will provide 1 virus particle for each cell on a plate, while an MOI of 10 represents ten virus particles per cell. However, several factors can influence the optimal MOI including cell line, cell type, transduction efficiency and gene of interest. We recommend first establishing an optimal MOI for each cell line. This can be done using a range of MOIs (0, 0.5, 1, 2, 5, 10, 50) to determine the MOI required to obtain optimal gene expression
A general Plasmid Amplification Protocol can be found here.
We recommend transforming abm’s plasmids into our ProClone™ Competent Cells (Cat. No. E003). The ProClone™ Competent Cells are high-efficiency chemically competent DH5α (E. coli) cells ideal for routine plasmid amplification due to its high yield, rapid growth, and high transformation efficiency, with recA⁻ and endA⁻ mutations that ensure plasmid stability and high DNA quality. Other common compatible cloning strains include TOP10 (DH10B derivatives).
1. Use a sterile loop or pipette tip to scrape a small amount of cells from the agar stab. Only a tiny amount is needed as it contains live E. coli. 2. Gently streak the cells onto an agar plate containing the correct antibiotic selection in order to achieve single isolated colonies. 3. Place the plate “agar side up” and incubate at 37°C overnight. 4. Select a single colony from the plate for downstream applications, such as an overnight broth culture and grow E. coli to late exponential-early stationary phase. 4a. The broth culture can be subjected to miniprep plasmid extraction. We recommend using abm’s Column-Pure Plasmid Miniprep Kit (Cat. No. G4003). 4b. The broth culture can be used to prepare a glycerol stock for long-term storage. Mix culture with sterile glycerol to a final concentration of 15% and store at -80°C.
We recommend first checking the following parameters: 1. Cell density. If density is too low, this can also result in cell death. We recommend the following experiments: Initial Experiment •Transduce lentivirus into the 293T cell line - one of the easiest cell lines to transduce. If the transduced cells survive selection, then troubleshooting should focus on post-virus production steps. If the transduced cells didn't survive selection, the troubleshooting should focus on virus packaging steps. Virus Packaging Troubleshooting •Attempt lentivirus packaging using a GFP expressing control vector. If packaging is successful with the control, then repeat packing of the lentivirus in question with a GFP batch control to ensure no errors with the first production. If no GFP signal is observed after transfection or ~72h after a test transduction of a GFP control lentivirus on 293T cells, then the packaging process needs to be assessed. Packaging process assessment includes reagent quality, cell health, and packaging protocol details. Post-Virus Production Troubleshooting •Assess if the correct antibiotic concentration is used. This can be done by performing a drug-killing curve. To set up a drug-killing curve, we recommend using the same culture size and seeding density for your actual selection, and adding a different puromycin concentration to each sample, with the range between 0-1 ug/ml. If the cells are not killed at the 1ug/ml, you may try increasing the range higher concentrations. It is important to identify the concentration that results in >95% cell death in 1-4 days to establish the minimal concentration to use for the selection process. •Assess if the correct MOI is used to transduce the target cells. This can be done by using a GFP control lentivirus to transduce the target cells at a range around literature-recommended MOIs to determine the optimal MOI for transduction. •Assess if a transduction enhancer such as the ViralEntry Transduction Enhancer (Cat.No. G515) is necessary. Some cell lines are more difficult to transduce than others and by using a transduction enhancer can lead to increased target cell permeability.
1. This often happens to primary cells which are very sensitive to culture medium conditions. Try to use a higher titer virus in order to limit the volume of exogenous media added to your cells. 2. Virus preparation might be contaminated with bacteria. Use a 0.25µm syringe filter to clear the virus preparation and repeat transduction following strict sterile technique. 3. Cell line might be contaminated with mycoplasm. This type of contamination is often not immediately obvious. The contamination effect is amplified after virus transduction. Repeat transduction with fresh cells. 4. The virus preparation may contain some cell debris which could be mistaken as a change in cell morphology. Normally this issue will disappear 3-5 days after virus transduction.
AAV exhibits natural tropism towards certain cells and tissue types. Therefore your choice of AAV serotype should be dependent on the desired cell type. See our AAV Serotype Selection Chart at this link. Alternatively abm offers an AAV Serotype Blast Kit (Cat. No. AAV099) containing 9 pre-packaging helper-free AAV with GFP expression. This kit can be used to help the user determine a suitable serotype for their desired cell line.
The sgRNA target sequences for all of abm’s CRISPR knockout vectors (human and mouse) can be found on each individual product’s webpage. Regarding rat specific genes, these sgRNAs are available upon order placement please email technical@abmgood.com
sgRNAs are primarily selected from widely trusted sources including Feng Zhang’s GEcKO v2 Knockout library or are designed using a variety of software to evaluate the best sgRNA targets including parameters such as high efficiency, minimal off targets and low self complementarity. We routinely find from in-house CRISPR knockout experiments that at least 1 out of 3 sgRNAs (from a set of 3 sgRNAs) results in successful gene knockout – this is why we always recommend choosing a set of 3 sgRNAs per gene.
There could many possible reasons for this result. 1. Typically GFP is behind a weaker promoter such as SV40 or CBH leading to a less robust fluorescence signal. 2. Virus titer is low leading to only a small proportion of successfully transduced cells. Try increasing the MOI or using a transduction enhancer such as abm’s ViralEntry™ (Cat.No. G516). 3. Target cells are difficult to transduce. A higher MOI must be tested. Some cells are very easy to be transduced such as 293T, while others such as lymphocytes are very difficult. 4. Lentivirus became inactivated during transportation and storage. Lentivirus is very sensitive to temperature changes. Leaving lentivirus at room temperature, 4°C for 48 hours, or subjecting it to multiple freeze/thaws can lead to inactivation or reduced titer.
There could be several possible reasons for this: 1.. Cells are a mixture of clones, not a single clone. We recommend isolating single cells by serial dilution and then screening and sequencing those cells. 2. CRISPR gene knock out efficiency is variable and dependent on many different factors (sgRNA, cell type, chromatin accessibility, expression levels of Cas9 etc) therefore it is recommended to screen 50-100 clones in order to identify positive edited clones. 3. We also recommend abm’s CRISPR Screening Kits (Cat.No. G990 and G932) to increase your accuracy and minimize false positives and negatives.
There could be several possible reasons for this: 1. Cells are polyclonal. Cells are a mixture of clones, not a single clone. We recommend isolating single cells by serial dilution and then screening and sequencing and testing those cells. 2. The cell line may not be diploid, but instead could be triploid or more. In other words, the cells have more than two copies of the same gene. This is a very common phenomenon in cancer cell lines. 3. The antibody being used may be binding to other proteins from the same family. Assess the antibody and ensure that it only binds to your target protein.
For SpCas9, the PAM sequence is NGG, and it is located on the non-target strand. The sgRNA binds the target strand (the strand complementary to the non-target strand sequence, excluding PAM).
As a general design rule:
Identify the PAM (NGG) on the non-target strand
The 20 nt protospacer immediately upstream of the PAM is the guide sequence used for sgRNA design
Cas9 cuts approximately 3 bp upstream of the PAM
When cloning sgRNAs, reverse-complement requirements depend on the specific cloning system and oligo design format; customers should follow the cloning instructions for the backbone being used.
This is a common issue and does not necessarily indicate the KO failed.
Common causes include:
(1) KO assessment performed too early GFP confirms transfection but does not guarantee functional KO at early time points. With transient systems, knockout efficiency may be limited at 24–48 hours. We recommend waiting 72–96 hours post-transfection before assessing KO outcomes. If possible, proceed with clonal isolation for the most reliable KO validation.
(2) Protein/mRNA stability (long half-life) Many proteins (and transcripts) have long half-lives, so residual signal may persist even after successful editing—especially in a pooled population where editing is heterogeneous.
Recommended KO validation workflow:
Enrich GFP⁺ cells (FACS recommended if available)
Extract genomic DNA from pooled cells
PCR amplify the target region
Perform Sanger sequencing and analyze indel frequency using ICE or TIDE
Proceed with single-cell cloning and validate KO clones using gDNA sequencing ± RT-qPCR / western blot / functional assay
Complete cell death during puromycin selection most commonly indicates one or more of the following: puromycin concentration is too high for the specific cell line or selection was initiated too early, before sufficient expression of the puromycin resistance gene. To optimize selection, abm recommends the following:
1. Perform a puromycin kill curve
Test a range of puromycin concentrations (e.g., 0.25–10 µg/mL, depending on the cell type).
Identify the lowest concentration that kills all untransduced control cells within 3–5 days.
Use a slightly lower concentration (typically 0.5×–0.75× of the kill-curve dose) for post-transduction selection.
2. Delay antibiotic selection
Allow 48–72 hours after transduction before adding puromycin.
This recovery period allows expression the puromycin resistance gene.
3. Confirm transduction efficiency
Include a positive control lentivirus (e.g., GFP-only or selectable marker–only) to verify infection efficiency.
4. Optimize infection conditions
Use an appropriate MOI for the target cell type.
Include a transduction enhancer (e.g., polybrene at 4–8 µg/mL, if compatible).
CRISPR gene knockout workflows can vary widely and typically require optimization for each experimental system. Outcomes depend on multiple factors, including cell type, target gene, delivery method, and selection strategy. As a result, there is no single universal protocol, and users should expect to optimize conditions for their specific application. General guidelines and example protocols are provided in the linked documentation to support experimental design and optimization.
Each experiment is unique and may require optimization depending on the cell type, but here is a helpful CRISPR Workflow Guide to point you in the right direction.
ABM Scientific Support
Answered on May 14 2026
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AAV serotypes 3, 4, and 6 typically produce lower yields
compared to other serotypes.
As such, we can only guarantee 50% of the minimum titers
listed in the table above.
AAV serotypes 3, 4, and 6 typically produce lower yields
compared to other serotypes.
As such, we can only guarantee 50% of the minimum titers
listed in the table above.