CRISPR/Cas9 Knockout Lentivirus Product: Lv-CBH-spCas9-EGFP                  Cat. No.: GMV-Crispr-LV007

CRISPR/Cas9 premade products

Cat No.	Products Name	Type of Crispr	Viral vector	Promoter	Fluorescent/Resistance	Tag	Order
GMV-Crispr-AAV001	AAV-CMV-saCas9	saCas9	AAV	CMV	Null	HA
GMV-Crispr-AdV001	Ad-CMV-spCas9	spCas9	Adenovirus	CMV	zsgreen	FLAG
GMV-Crispr-AdV002	Adv-CMV-spCas9-zsgreen	spCas9	Adenovirus	CMV	EGFP	FLAG
GMV-Crispr-AdV003	Adv-CMV-spCas9-EGFP	spCas9	Adenovirus	CMV	Null	FLAG
GMV-Crispr-AdV004	Adv-CMV-spCas9-mcherry	spCas9	Adenovirus	CMV	mcherry	FLAG
GMV-Crispr-LV001	Lv-CMV-spCas9-puromycin	spCas9	lentivirus	CMV	puromycin	FLAG
GMV-Crispr-LV002	Lv-CMV-spCas9-zsgreen	spCas9	lentivirus	CMV	zsgreen	FLAG
GMV-Crispr-LV003	Lv-CMV-spCas9-EGFP	spCas9	lentivirus	CMV	EGFP	FLAG
GMV-Crispr-LV004	Lv-CMV-spCas9-mcherry	spCas9	lentivirus	CMV	mcherry	FLAG
GMV-Crispr-LV005	Lv-CBH-spCas9-puromycin	spCas9	lentivirus	CBH	puromycin	FLAG
GMV-Crispr-LV006	Lv-CBH-spCas9-zsgreen	spCas9	lentivirus	CBH	zsgreen	FLAG
GMV-Crispr-LV007	Lv-CBH-spCas9-EGFP	spCas9	lentivirus	CBH	EGFP	FLAG
GMV-Crispr-LV008	Lv-CBH-spCas9-mcherry	spCas9	lentivirus	CBH	mcherry	FLAG

Custom made CRISPR/Cas9 service

Custom-made CRISPR/Cas9-gRNA lentivirus for knockout cell line development

Custom-made CRISPR/Cas9-gRNA adenovirus for knockout cell line development

Custom-made CRISPR/Cas9-gRNA AAV for tissue-specific knockout in vivo

CRISPR/Cas9 User Manual

Crispr/cas9 mediated Gene knockout

AAV Production CRISPR/Cas9 Knockout System-User Manual

Adenovirus CRISPR/Cas9 Knockout System-User Manual

Lentivirus-CRISPR/Cas9 Knockout System-User Manual

With Genemedi's CRISPR/Cas9-gRNA lentivirus packaging service,scientists can easy to achieve multiple gene-knockout in different cell lines.Genemedi can also supply unique gene knockout service in specific cell line demanded with crispr/cas9 gene editing tool.

HIV-1 (human immunodeficiency virus type I) based defective lentivirus has been one of the most widely used gene therapy vectors. It is a powerful tool for introduction of exogenous genes. The most advantageous feature of lentivirus vectors is to mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells. The lentivirus system has been widely used in various cell lines for gene overexpression, RNA interference, microRNA research and in vivo animal experiments.

CRISPR/Cas9 gene editing technology has revolutionized the field of genome modification, using two key components that form a complex: Cas9 endonuclease and a single guide RNA (sgRNA) that guides Cas9 to a specific target site in the genomic DNA. It was listed as one of the top 10 breakthrough discovery in 2013. The technology has (1) higher targeting accuracy; (2) much more target sequence selection; (3) much less complexity; and (4) much less off-target cell toxicity than the previous genome editing technologies: TALEN (transcription activator-like effector nuclease) and ZEN (Zinc-finger nuclease).

The Genemedi CRISPR/Cas9 System is a complete system for producing high yields of lentiviruses encoding the components necessary for CRISPR/Cas9-mediated genome editing [i.e., single guide RNA (sgRNA) and Cas9 nuclease] for delivery to mammalian cells that are difficult to transfect.

1. High efficient Cas9 expression delivery with markers : High titer lentivirus providing more efficient Cas9 delivery in almost all cell 9 types including primary cells and non-dividing cells; Some Cas9 products include a fluorescent-antibiotic dual marker allowing the real-time check the lentivirus transduction efficiency.

2. Best nuclear penetrating for Cas9 enzyme: the Cas9 is expressed with an optimized, proprietary Nuclear Localization Signal (NLS), providing the efficient cas9 delivery into the nuclear region where the gene editing occur.

3. No need for tedious cloning work or vector construction: you can simply synthesize the gRNA (and donor cassette when desired) and used together with the Cas9 lentivirus for the gene editing.

4. Allow multiple gene editing at the same time: no need to construct each targeting vector for different gene. Instead, you just select the target sequence and synthesize the gRNA (each single strand RNA or double stranded DNA cassette) that to used with ourCas9 expression particles.

Our optimized custom lentiviral vector production and strict quality control systems provide customers a high titer of functional recombinant lentiviral vectors. Viral titers are determined by two methods: functional (infectious) titer (TU/mL) and physical titer (VP/mL). Physical titer is calculated by the level of protein, such as p24, or viral nucleic acid. The functional titer, a calculation of the active virus that can infect cells, is much less than the physical titer (100-1000 fold lower). Direct functional titer is an accurate solution for testing the MOI but is time consuming and not feasible. The physical titer is sufficient for most lentiviral experiments. Our main titration procedure is to determine the quantity of physical lentiviral particles. We can also calculate the functional lentiviral vectors with additional cost if required by customers.

1. For further information about lentivirus administration and transduction, please see Lentivirus User Manual.

2. For further information about autophagy study, please see CRISPR/Cas9 Knockout Lentivirus User Manual.

1.What is “CRISPR”?

Answer

“CRISPR” stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term “CRISPR” or “CRISPR-Cas9” is often used loosely to refer to the various CRISPR-Cas9 and -CPF1, (and other) systems that can be programmed to target specific stretches of genetic code and to edit DNA at precise locations, as well as for other purposes, such as for new diagnostic tools.

2.How does the system work?

Answer

CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the gene’s function.

3.How does CRISPR-Cas9 compare to other genome editing tools?

Answer

CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research community. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which is another advantage that sets it apart from other gene-editing tools.

4.How does CRISPR-Cpf1 differ from CRISPR-Cas9?

Answer

CRISPR-Cpf1 differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics.

First, in its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

Second, and perhaps most significantly, Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving ‘blunt ends’ that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.

Third, Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur.

Fourth, the Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting, for example, the malaria parasite genome and even the human genome.

5.What other scientific uses might CRISPR have beyond genome editing?

Answer

CRISPR genome editing allows scientists to quickly create cell and animal models, which researchers can use to accelerate research into diseases such as cancer and mental illness. In addition, CRISPR is now being developed as a rapid diagnostic. To help encourage this type of research worldwide, Feng Zhang and his team have trained thousands of researchers in the use of CRISPR genome editing technology through direct education and by sharing more than 40,000 CRISPR.

6.How to design the 20bp target-specific sequence?

Answer

The 20bp target-specific sequence should precede NGG (PAM). Please BLAST the seed region (8-14 bp PAM-proximal) of the 20bp target sequence to make sure it’s unique along the genome to guarantee its specificity.

7.How to avoid off target issue using CRISPR/Cas?

Answer

You can blast your target sequences. If the off-target sequences don’t have the PAM (NGG), then they won’t be targeted by CRISPR/Cas9. You also want to choose target sequences with mismatches in the 8-14 bp at the 3’ end of the target sequences. This way, the off-target issue can be decreased dramatically. For therapeutic purpose, you can use Cas9 nickase which only cuts one strand.

8.How many target RNA sequences should I use for a genome editing project?

Answer

Due to the un-predicable nature of gRNA, we recommend 3 and more gRNA targeting sequences to be designed to make sure that at least one targeting sequence will provide efficient cleavage.

9.Do you know the specific cleavage site of the Cas:gRNA complex in terms of where in the targeting sequence the cleavage occurs?

Answer

Cas9 cleaves at 3 bp away from the 3’ end of the target sequence in the genome.

10.Why I cannot find the gRNA targeting sequences in the cDNA sequence?

Answer

The targeting sequences could be located in either exon or intron in the genome; the cDNA sequences only contain the exons. CRISPR/Cas9 will target the genomic sequence, then genome editing will be achieved.

11.The transfection efficiency of my cell line is only 20%, how to enrich CRISPR transfected cells?

Answer

You can use pCas-Guide-EF1a-GFP to enrich transfected cells since GFP is also expressed. We also have pCas-Guide-EF1a-CD4 vector; you can use anti-CD4 antibody beads to enrich transfected cells. Alternatively, you can transfect a plasmid with a selection marker and select the cells.

12.How to screen the edited cells after transfecting the CRISPR/Cas9 vector?

Answer

For mutations, you can do genomic PCR and sequence it. If you do gene knockout, the selection marker in the donor template DNA will help the selection. If no donor DNA for gene knockout out, then genomic PCR and sequencing to confirm indels. If necessary, you can isolate individual cell colonies for introduction of specific mutations and other genome editing applications. You can do WB for gene knockout after isolating single cell colonies.

13.Does CRISPR/Cas system work for non-dividing cells?

Answer

NHEJ repair works in non-dividing cells; HDR is not active in non-dividing cells.

14.Using CRISPR, can you get monoallelic knockout (heterozygous) or biallelic knockout (homozygous)?

Answer

CRISPR/Cas9 double-strand cleavage is very efficient. If just using CRISPR/Cas9 vectors to introduce indels, if transfection efficiency is high, more biallelic knockout can occur. In the presence of donor DNA, since homologous recombination may be a limiting factor, some cells contain monoallelic knockout and some cells contain biallelic knock out.

15.If I want to use CRISPR/Cas9 to knock down a certain gene, what kind of negative control should I use?

Answer

You can use a scramble control, pCas-Scramble, SKU GE100003, or pCas-Scramble-EF1a-GFP, SKU GE100021.

16.How long does the service take? Is my project too big (or too small)?

Answer

Get ready-to-transduce, high-quality, high titer lentiviral preparations at the production scale your project needs—even at large scales of up to 10 mL. Use your own lentivector construct or take advantage of our Custom Construct services and we’ll handle vector construction as well. We even have an ultra-high titer offering for demanding applications such as in vivo and stem cell transductions.

16.For gene targeting in mice, do you recommend transfecting ES cells or pronuclei?

Answer

You can do both. You can inject mRNA (gRNA and Cas9 mRNA) or plasmid DNA (target sequence cloned pCas-Guide) into the zygotes or ES cells.

17.How do you make sure that Cas9 will not integrate in genome if you use lentivector?

Answer

For screening purpose, for short term, integration of Cas9 into the genome for 2 weeks does not affect cells.

18.Do you see variability in success with different cell lines?

Answer

Yes, depending on the cell line and the gRNA sequences.

19.What is the known CRISPR/Cas9 editing efficiency relative to other genome editing approaches?

Answer

In general, the genome editing efficiency of CRISPR/Cas9 is similar or higher than TALEN. However, CRISPR/Cas9 is much more simple and easy to do. You will need to engineer the protein to recognize new DNA sequence in TALEN system, while CRISPR/Cas9 is RNA based.

20.Is there any safety issue with this pLenti vector?

Answer

The pLenti vector is a third generation lentiviral vector and it is the safest lentiviral vector because both LTRs are truncated. Please contact the biosafety office at your institution prior to use of the pLenti vector for permission and for further institution-specific instructions. BL2/(+) conditions should be used at all times when handling lentivirus. All decontamination steps should be performed using 70% ethanol/1% SDS. Gloves should be worn at all times when handling lentiviral preparations, transfected cells or the combined transfection reagent and lentiviral DNA.

21.Do I get monoallele knock-out or biallele knock-out using the homology-mediated knock-out kit via CRISPR? What do I need to do to get biallele knock-out?

Answer

If you isolate single cell colonies, in some cells gene knock-out may occur only in one allele; in some cells gene knock-out may occur in both alleles. If you only have monoallelic knockout and you want to get biallelic knockout, you can order another donor vector containing a different mammalian selection marker, such as blastocidin or neomycin resistant marker. Make sure the other allele is intact as it can be targeted and repaired via NHEJ; confirm with genomic PCR and sequencing. You can do the knockout procedure again with the new donor vector to target the second allele (one allele is already targeted and replaced with GFP-puro cassette).

22.What could be the reason that I couldn’t get my gene of interest knocked out?

Answer

If your target gene is essential for cell survival, you might not be able to get constitutive gene knockout. Conditional gene knockout may be needed.

23.Can I use the nickase instead of wild-type Cas9?

Answer

Yes. We have the reagents for the Cas9 D10A nickase, and have successfully tested our double nickase designs. However, in order to create mutagenic DSBs, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. Because proper sgRNA targeting requires the presence of the N-G-G “PAM” site immediately following the recognition site, it might not always be possible to use the nickase for DSB formation. There are also “high-fidelity” variants of Cas9 nuclease that edit genes with greater specificity than wild type Cas9, but sometimes with reduced efficacy and with increased design constraints. However, since these high fidelity variants use only one sgRNA, they are easier to work with than Cas9 niclases.