Hi Christian,

as far as I know from the literature, the homology arms should be as close to the cut site as possible. Definitely within 100 bp, ideally less than 10 bp away.

You can also check here for more information

https://www.addgene.org/crispr/zhang/FAQ/

especially, HR FAQs point 2 addresses your question directly. Importantly, you should also definitely mutate the PAM sequence in your repair template to avoid its cutting by Cas9.

Hope this helps and best of luck!

Hi Christian,

The reason for not getting the knockin is most likely because your are using a double stranded plasmid as HDR template. 

We published recently that you can get very high efficiencies by using a single stranded HDR template, generated with rAAV.

http://www.ncbi.nlm.nih.gov/pubmed/25586224

Best,

Manuel

 hi,Christian , How was your experiment going on? I am doing the same thing, I want to know how did you solve the problem. 

hi,

I want to construct HDR-repair template  donor vector  to knock-in GFP at the targeted gene site  in Arabidopsis thaliana plant. can any one suggest me how can i construct and which type of vector backbone can i use for this.

you may use Benchling tool to design HDR template for any gene of your interest,determining you gRNA and PAM as well as point of restriction, then you can use this backbone plasmid on addgene as a vector for your designed HDR template arms 

https://www.addgene.org/63934/

can anyone help me out that for knockin it is necessary to have LR and RB in donor vector and second what is the insertion fragment length for knockin exp.

Are you applying a continues selection pressure for homoplasm?

no, I want transient expression via particle bombardment...

agree with @ Manuel Kaulich

regards

CRISPR donor vector is very specific for your experiment purpose. Few big company will offer the service. Your can try http://www.noahgen.com/ for a customized cloning.

Improving lentivirus titer requires a multifaceted approach to optimize each step of the virus production process:

1. Optimize Transfection Efficiency: Use high-quality transfection reagents and optimize the transfection protocol for your specific cell line to ensure maximal transfection efficiency.

2. Cell Density: Harvest cells at optimal confluency; overgrown or sparse cells can lead to reduced viral production.

3. Quality of Plasmid DNA: Use a high-quality plasmid preparation with minimal endotoxin levels to avoid cytotoxicity and ensure optimal expression of viral proteins.

4. Third-Generation Packaging System: Utilize a third-generation packaging system which separates the viral components into multiple plasmids to enhance safety and titer.

5. Ratio of Plasmids: Fine-tune the ratio of the lentiviral packaging plasmids to the transfer vector; often a slight excess of the packaging plasmids can increase titer.

6. Serum Quality: Use high-quality fetal bovine serum (FBS) in your cell culture media during virus production, as serum quality can greatly affect viral production.

7. CO2 and Temperature: Ensure your incubator is correctly calibrated for CO2 and temperature, as deviations can affect cell health and viral production.

8. Harvest Timing: Collect the viral supernatant at the optimal time post-transfection when viral production peaks, usually between 48 to 72 hours.

9. Concentration Technique: After collection, concentrate the lentivirus using ultracentrifugation or filtration methods to increase the titer.

10. Storage Conditions: Store the virus at -80°C in aliquots to prevent freeze-thaw cycles that can decrease the titer.

11. Titer Assa: Regularly perform titer assays to quantify the improvements in your lentivirus production and adjust the protocol accordingly.

By systematically optimizing these factors, you can significantly improve the titer of your lentiviral preparations.

This protocol list might provide further insights to address this issue.

Designing a donor template for homology-directed repair (HDR) in CRISPR applications involves precise construction to ensure successful integration. Here’s a guideline:

1. Target Sequence Identification: Identify the genomic location where you wish to insert the donor DNA. This should be in close proximity to the CRISPR-Cas9-induced double-strand break site.

2. Homology Arms: Design homology arms that are typically 200-1000 base pairs in length on either side of the insertion site. These arms should be identical to the sequences flanking the cut site to facilitate recombination.

3. Insert Design: Include the sequence you want to insert within the homology arms. This could be a point mutation, a tag, a reporter gene, or any other sequence.

4. Avoiding CRISPR Cut Sites: Ensure that the donor template does not contain PAM sequences or sequences highly similar to the gRNA binding site to prevent the Cas9 from cutting the donor DNA.

5. Vector Backbones: Clone the homology arms and the insert into a suitable vector that facilitates the amplification and purification of the donor template.

6. Testing in Silico: Use bioinformatics tools to simulate the HDR process and check for potential off-target insertions or recombination events.

7. Verification: Sequence the entire donor template to confirm that it has been constructed correctly without any errors or unwanted mutations.

8. Transfection Efficiency: Use a transfection method with high efficiency for your cell type to deliver both the CRISPR-Cas9 components and the donor template into the cells.

9. Screening for HDR: After transfection, screen for HDR events using PCR, sequencing, or functional assays, depending on the nature of the insert.

10. Optimization: Be prepared to optimize the design based on empirical results, as HDR efficiency can vary greatly depending on the target cell type, the genomic context, and the nature of the insert.

Reviewing the protocols listed here may offer further guidance in addressing this issue.