HDR Design Tool FAQ

Technology Questions

• What is Homology Directed Repair (HDR)? How is it used in CRISPR-mediated knock-in experiments?

  Homology Directed Repair (HDR) is a DNA repair mechanism that occurs naturally in cells. It is a process that uses a template DNA sequence to repair a double-strand break (DSB) in the DNA molecule. HDR relies on the presence of a homologous DNA sequence to guide the repair process.

  To utilize HDR for gene editing, researchers typically introduce a custom-designed DNA template (HDR template) into the cell along with CRISPR-Cas9. The HDR template sequence is designed to carry a left homologous arm, a target sequence you want to knock in, and a right homologous arm. The cell's repair machinery uses this template to repair the DNA break, resulting in the desired genomic insertion.

• What are the advantages of non-viral CRISPR-mediated knock-ins?

  Compared to viral-based CRISPR delivery, non-viral delivery offers relatively low immunogenicity, simple and fast manufacturing, high packaging capacity, and transient editing- reducing the risk of off-targeting.

• How do GenScript HDR templates compare to traditional plasmid templates for CRISPR-mediated gene knock-ins?

  Assuming the target application is ex-vivo cell engineering via non-viral delivery, GenScript HDR templates offer the follow advantages over traditional plasmids:

  • GenExact ssDNA and GenWand dsDNA are linear templates which is easier for HDR process and has lower cytotoxicity than traditional plasmid templates.
  • GenCircle dsDNA has a circular template that is much smaller in size compared to a traditional plasmid. This helps to decrease cell cytotoxicity by up to 33% and increase KI efficiency by up to 30%. GenCircle dsDNA also has no antibiotic resistance gene, which helps to avoid drug resistance concerns and regulatory concerns.

Design Tool Protocol

• What are the design capacities of GenScript's HDR knock-in design tool?

  Our HDR knock-in design tool can help you:

  • Design sgRNA and HDR templates for 150nt to 20kb according to your target gene in specific sites for different species.
  • Design an HDR template according to your target gene and specific sgRNA.

  You can optimize your HDR template by:

  • Adding different tags for detection, purification, or localization of the target protein.
  • Choosing a custom homology arm length.
  • Adding a silent mutation to avoid a second cut by the Cas9/sgRNA RNP.
  • Adding a CTS sequence to increase your knock-in efficiency by up to 20% - 40%, according to reference

  We will provide the detailed parameter of different design results as a reference for you to choose. You can also decide the type, quantity, purification methods and delivery formation of your desired HDR template or sgRNA.

• Can you do Cas Nickase or cpf1 based KI design?

  Currently, our HDR knock-in design tool only supports Cas9-based designs. However, for nickase- or Cas12a/Cpf1-based knock-ins, our technical support managers can help guide you through your design off-line and we can provide the necessary guide RNA and HDR templates. Please contact crispr@genscript.com to get started.

• How do I use the HDR design tool?

  The general process for designing an HDR template includes the following steps:

  • Enter target gene information: Choose your target species, enter your target gene (either by gene symbol or gene ID), and choose Transcript ID.

  Optional steps:

  • Enter the edit location in number or sequence if you prefer a specific edit location.
  • Enter 19-20 guide RNA sequence (exclude PAM) if you prefer to use a specific guide RNA.
  • Choose suitable HDR template parameter: Select the sequence you want to replace by moving the green label and enter the sequence you want to knock in in the mutation box. Choose your desired tags (please remember click insert button to add tags), homology arm length, and silent mutation preference. We have detailed sequence and translation information in the picture for your reference.
  • Select desired sgRNA & HDR template: Choose one or several sets of sgRNA and HDR templates according to your needs. Alternatively, you can choose an sgRNA or HDR template only. The “detail” option can be clicked to display and compare off-target scores. A CTS design can also be added to ssDNA or dsDNA to increase knock-in efficiency after your desired HDR template is chosen.
  • Choose specifications for sgRNA & HDR template: Select the quantity, purification methods, and delivery formation of your desired HDR template or sgRNA and add to cart. For ssDNA with a CTS design, you will also need to add annealing oligos for your experiment.
  Watch How to Video
• How can I change the position or length of my edit and explore the sequence map?

  Select editing position:

  • The green label represents the targeted editing site.
  • When the mouse is placed on both ends of the green label and changes into a cross shape, press and hold the left mouse button to adjust the position on the left and right ends of the green label, and select a suitable editing position.

  Go trough sequence map:

  • By placing the mouse cursor on the sequence map and scrolling the mouse wheel or clicking “Zoom to edit” button, you can zoom in on the sequence map to display each base and amino acid.
  • Press and hold the left mouse button to move the sequence map freely to the left or right.
• What length should I use for the homology arms? If I choose the default, how are the homology arms chosen and position on my HDR template?

  Based on internal testing, we recommend the following homology:

  40-50nt HA is good enough for point mutations and a few nt insertions and deletions. For a longer insertion size (<100 nt), 70 nt HA is acceptable. For insertion sizes larger than 100 nt, longer HA (>=300nt) is better. However, knock-in efficiency may decrease as the insert size increases.

  Insertion length ≤2kb:

  • ssDNA: homology arm is recommended as 250nt
  • dsDNA: homology arm is recommended as 150bp-200bp

  Insertion length＞2kb:

  • homology arm is recommended as 300bp-500bp

  Default homology arm in the design tool:

  • 70nt HA for ≤100bp inserts
  • 300nt HA for >100bp inserts
• What is the purpose of adding silent mutations to my HDR template design? How do I add a silent mutation to my template using your design tool?

  Adding silent mutations to your HDR template can enhance the precision and accuracy of gene editing.

  Cas enzymes can continue to cut DNA until the gRNA and PAM sites are disrupted and the new DSB will be repaired through NHEJ or HDR again. Adding silent mutations help ensure that the desired edits in the gene editing process remain intact by preventing the Cas9 nuclease from re-cutting the edited site.

  To add silent mutations to your template in our design tool, simply check the 'add silent mutations' box after entering your mutation.

• How are silent mutations added in GenScript's HDR design tool? How do you modify the sequence?

  GenScript's HDR design tool has a Add silent mutations option for silent mutations function. If it is chosen, tool will check gRNA target exists on the arm of donor template and make a silent mutation (mutation that changes only the DNA sequence with the help of synonymous codons) in the region where gRNA found if necessary when design donor template sequence. PAM and the region close to PAM are the referred regions for silent mutation.

• What tag options does the tool offer?

  Tags are commonly added to HDR templates in CRISPR-mediated knock-in experiments. While they can serve multiple functions, their primary uses include purification, detection, or localization of the target protein. You can choose from the following tag types in our HDR template design tool:

  Affinity tags: facilitate the detection and purification of proteins, increase the yield of recombinant proteins, enhance the solubility of recombinant proteins, and promote the proper folding of recombinant proteins. We offer:

  1. His: protein purification
  2. GST: protein purification, immunoassay, enzymatic analysis
  3. HiBit: quantification & detection of proteins in live cells, bioluminescence

  Epitope tags: allow a protein to be detected by antibodies. Epitote tags are widely used in western blotting, immunoprecipitation, and immunofluorescence. We offer:

  1. FLAG: Western Blot, IHC/ICC/IP, Flow cytometry, ELISA
  2. Myc: Immunoprecipitation, protein purification, flow cytometry
  3. HA: purify tagged protein, western Blot, IHC/ICC/IP, flow cytometry, ELISA
  4. V5: Western Blot, IHC/ICC/IP, Flow cytometry

  Fluorescent tags: used to label the expression of proteins and can be used in live cells. Fluorescent tags are first brought to an excited state by exposing them to ultraviolet or blue-violet light. When they return to the ground state from the excited state, they emit fluorescence. We offer:

  • eGFP: live fluorescent imaging, flow cytometry

  Protein degradation tags: regulate the protein degradation system in cells and clear target proteins. We offer:

  • dTag: degrade target proteins

  Other:

  • Kozak: Enhance transcription and translation efficiency in eukaryotes.
• What are GS linkers?

  GS linkers are short DNA sequences recommended to be added between the tag and the endogenous protein-coding sequence to minimize the functional disruption of the target protein. Commonly used GS linker sequences are composed of amino acids Gly and Ser in different numbers and combinations, e.g., GSGSGS.

  We recommend to choose 3x GS linker (18 bp) for insertion ≤100 nt; 5X GS linker (30 bp) for insertion >100 nt).

• How many sets of sgRNA and HDR should I order for my experiments?

  We recommend testing at least 3-4 designs.

• I already have a donor sequence that I want to use for my knock-ins. Can I use your HDR template design tool to order a DNA payload containing my sequence?

  Yes, you can easily order DNA payloads in a ssDNA, linear dsDNA, or circular dsDNA format using your own sequence. Simply add your sequence to our GenCRISPR DNA Payload online ordering system to get started.

• What should I do if I can't find my target gene?

  Please double check that the information you entered is correct. If you still can’t find your gene, please contact us for technical support (crispr@genscript.com).

• What should I do if I can’t find a suitable silent mutation site or can’t find suitable sgRNA cleavage sites within the 30bp range of the target edit site?

  Please try again with a different edit site or contact us for technical support (crispr@genscript.com).

• What should I do if there is no suitable silent mutation?

  Please contact us for technical support (crispr@genscript.com).

• What is CTS? How does it work? How to add CTS to my HDR templates? What is annealing oligo? Why is it needed?

  In addition to standard HDR donor DNA designs, we offer the option to add a Cas9 Targeting Sequence (CTS) to your ssDNA and dsDNA designs to improve gene knock-in efficiency. A CTS allows for the co-delivery of the donor DNA and sgRNA/Cas9 complex into the nucleus target gene site, increasing the HDR knock-in rate. However, CTS design is not suitable for circular HDR template such as GenCircle dsDNA. More info on CTS design can be found in this paper.

  If selected, a CTS is added to the 5’ of the ssDNA to improve knock-in efficiency. Data from our collaborator, Dr. Brian Shy, have shown that CTS at 5’ alone can drive an improved knock-in rates.

• Do I need to add CTS to all my HDR templates?

  No, it is not mandatory to add CTS designs to all the HDR templates. CTS design is not suitable for circular HDR template such as GenCircle dsDNA. However, it is recommended to add the CTS sequence to the 5' end of the ssDNA Or dsDNA HDR template due to the reported benefits of increasing knock-in efficiency.

• What should I do if the tool failed to generate designs? What should I do if the HDR templates have errors?

  Design failures or errors may indicate that there are no suitable sgRNA cleavage sites within the 30bp range of your requested cleavage site. If you experience either of these scenarios, please try again with a different edit site or contact us for technical support at crispr@genscript.com.

• How can I go back to the previous step to check the information entered or make a modification?

  You can get back to the previous step in our design tool by either clicking the 'back' button in the bottom of page or by clicking the process name in the upper right corner of webpage.

• What is the maximum HDR template length that you can chemically synthesize?

  Here is the standard service information and also the length range in GenScript HDR knock-in design tool:

  • GeneExact ssDNA: 150 nt - 5 kb
  • GenWand dsDNA: 1-10 kb
  • GenCircle dsDNA: 1-20kb

  Request for HDR template lengths outside the ranges listed above may be accepted and are evaluated on a case-by-case. Please contact us for technical support at crispr@genscript.com.

Service Information

• How do I know which HDR template format is best for my CRISPR-mediated gene knock-in? What are the applications of HDR templates (GenExact ssDNA, GenWand linear dsDNA, and GenCircle circular dsDNA)?

  We offer three HDR template formats: GenExact ssDNA, GenWand linear dsDNA, and GenCircle circular dsDNA. Based on our experience and customer feedback, knock-in rates highly depend on the cell type, insert size, delivery protocols and template length. For this reason, we recommend testing out multiple types of HDR template formats and comparing their performance in your specific experiment. However, these are our general guidelines:

  • GenExact ssDNA: 150-5,000 nt, minimal cytotoxicity, precise knock-in, minimized off-target effect, ideal for T cell therapy
  • GenWand dsDNA: 1-10 kb, covalently closed ends protect for better accuracy, more suitable for scale up
  • GenCircle dsDNA: 1-20 kb, small circular template with no resistance gene, only 429bp backbone, lower cytotoxicity & higher transfection efficiency, ideal replacement for plasmid HDR templates
• What are the applications of HDR templates (GenExact ssDNA, GenWand linear dsDNA, and GenCircle circular dsDNA)?

  Here are some examples of applications for our HDR templates:

  GenExact ssDNA:

  • Genome Editing
  • Non-viral gene therapy
  • Animal model/ cell line generation
  • In vitro diagnostic
  • Structural DNA nanotechnology (Scaffolded DNA origami)

  GenWand dsDNA:

  • Genome Editing
  • Non-viral gene therapy
  • Animal model/ cell line generation

  GenCircle dsDNA:

  • HDR knock-in template or transposon vector for CAR/TCR for cell therapies
  • HDR knock-in template or transposon vector for gene function/disease modeling studies
  • AAV/LV packaging plasmid, non-viral GOI vector, or mRNA preparation template for gene therapies
  • Antigen expression vector for DNA vaccines
• What’s the different between EasyEdit sgRNA and SafeEdit sgRNA? Which type should I choose?
  • EasyEdit sgRNA is purified by an optimized desalt method which is effective in commonly used cell lines. Fast delivery and competitive pricing makes it ideal for screening or early stages of research.
  • SafeEdit sgRNA is purified by HPLC, resulting in a guaranteed minimum of 90% purity. This option is ideal for the validation stage of development. Additionally, HPLC grade sgRNA is recommended for more challenging cell types such as stem cells and primary cells.
• What grades do you offer for your HDR templates? Do you have GMP? Which type should I choose?

  We offer RUO, GMP-like and cGMP grade HDR templates in all three formats (GenExact ssDNA / GenWand dsDNA / GenCircle dsDNA). The graphic shown below can be used to find the suitable grade for your development phase.

  
• What is the maximum quantity that can be ordered for an HDR template?

  We generally provide μg to g level GenWand dsDNA / GenCircle dsDNA and μg to mg level GenExact ssDNA. For quantity requests outside these ranges, please contact us for technical support at crispr@genscript.com.

• What container and delivery formats are available?

  Our HDR templates and sgRNA can be delivered in either single tubes or 96-well plates, in the following formats:

  • HDR templates (GenExact ssDNA / GenWand dsDNA / GenCircle dsDNA): either liquid and freeze dried
  • sgRNA: freeze dried

Experiment Questions

• What is the best method for measuring KI efficiency with my HDR template in my CRISPR-mediated experiments?

  It depends on the aim of the experiment: 1) point mutation or small fragment insertion, design primers that are flanking the modification site, PCR the edited cell’s genomic DNA, and then send for Sanger sequencing for verification; 2) fluorescent protein gene insertion, check the edited cells on flow cytometry with the right channel for fluorescent protein, eg. FITC for GFP; 3) CAR/TCR or other large fragment sequence insertion, please prepare the corresponding antibody for flow cytometry detection in advance of gene editing design work.

• How can I achieve high knock-in efficiency in my CRISPR-mediated experiments? What should I do if I detect low HDR efficiency in transfected cells?

  The following factors should be considered when optimizing your process to achieve better knock-in efficiency:

  Design stage:

  1) Appropriate knock-in site: the distance between knock-in site and DSB should not exceed 100 bp. The ideal distance is within 10 bp [2]. Select the PAM site that is closest to the desired insertion site, as it will likely have the highest cutting efficiency. Normally, the distance between the knock-in site and DSB will be within 30 bp in GenScript HDR design tool. We will show the distance in the design results parameter for your reference.

  2) HDR template type: GenExact ssDNA supports 150-5,000 nt knock-ins. For longer knock-in sequences, we suggest GenCircle dsDNA or GenWand dsDNA. You can also try different types of HDR templates to find the most suitable option for your experiment.

  3) Length of homology arms (HA): 40-50nt HA is good enough for point mutations and insertions and deletions of a few nt. For a longer insertion sizes (<100 nt), 70 nt HA is acceptable. For insertion sizes larger than 100 nt, we recommend longer HA (>=300 nt).

  4) Silent mutations: To prevent Cas9 recognition and cleavage of the donor after insertion into the genome, synonymous mutations can be introduced into the sequence corresponding to the PAM to change the PAM sequence [2].

  5) Other design strategies: A modification on the 5’ end of the HDR template (dsDNA) [3], or a CTS design in one or both ends of the HDR template [1] can be utilized.

  Experiment stage:

  1) Select an appropriate cell line.

  2) Try chemosynthetic sgRNA and Cas9 protein instead of plasmid sgRNA and Cas9. Choose an sgRNA sequence with robust editing activity.

  3) Test and choose HDR templates at a concentration with less cytotoxicity.

  4) Optimize reagent concentration and electroporation conditions. You can download one of our CRISPR knock-in protocols for reference or use our CRISPR non-viral CAR-T Knock-In Optimization Kits to optimize the experiment system first.

  5) Use a knock-in enhancer.

  [1] High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nature Biotechnology

  [2] Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol.

  [3] An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms. Nature chemical biology.

• Does GenScript provide modifications to HDR templates?

  We can provide modifications according to your request. Please contact us for technical support at crispr@genscript.com.

• How should I store my sgRNA and HDR template?
  • sgRNA: Dissolve the powdered sgRNA in TE buffer to a concentration of 100 pmol/µL, or other desired concentration. Divide into aliquots as needed and store at -20℃, avoiding repeated freeze-thaw cycles. For long-term storage, store at -80℃.
  • GenExact ssDNA/GenWand dsDNA: Keep the ssDNA/dsDNA tube(s) tightly sealed at -20°C for long-term storage and avoid repeated freeze-thaw cycles after dissolving. Centrifuge tube(s) at 12000 rpm for 3 minutes before opening to ensure that samples are at the bottom of the tube(s). If necessary, divide the stock solution into small aliquots. Stock solutions with a high concentration (> 100 ng/μL) can be stored at -20°C for up to 6 months.
  • dsDNA: Lyophilized plasmid is stable under -20°C for at least one year. Liquid plasmid products need to be stored at -20°C and avoid repeated freezing and thawing.
• How can I generate conditional alleles in mice?

  While CRISPR/Cas9-mediated production of null alleles in mice is highly efficient, the generation of conditional alleles has proven to be more difficult. Here are a few tips to help set you up for success:

  1) HDR efficiency can be improved by carefully designing and choosing sgRNAs, as they are highly correlated with Cas9 cutting efficiency.

  2) Use long ssDNA, rather than dsDNA, for significantly reduced off-target integration.

  3) While generating conditional alleles via the integration of LoxP sequence, using one long ssDNA is more efficient than using two short ssDNAs.

• How can I make sure to obtain biallelic knock-in in my model?

  If it is for transgenic model generation, please do sequence verification and select the right clone.

• How should I prepare the HDR donor template for experiments?
  • Centrifuge all samples before opening to ensure that oligos are at the bottom of the tube(s).
  • Use nuclease-free water or other desired buffer to dissolve the DNA template into the stock or working concentration. When dealing with mammalian cell lines, embryos, or primary cells, the buffer should be free of endotoxin and microorganisms.
  • To open potential secondary structures, heat the ssDNA/dsDNA sample at 70°C for 5 minutes and the store on ice before use.
  • When using a working concentration of 1 μg/μl or higher, pipet the solution multiple times to dissolve all samples evenly. Then, another round of centrifugation is recommended to ensure that the samples are at the bottom of the tube(s).
  • Store at -20°C for long term storage.
• How much sgRNA and HDR template should I order for 3 sets of transfection or X x10^6 of my cells (iPSC, immortalized cells, primary T cells?)
  • 293T cell: 2 x 10^5 cell / well, add 22.5 pmol sgRNA and 2 μg-4 μg ssDNA/ 0.5 μg-3 μg dsDNA per well. (For HDR template lengths between 0.5-4 kb)
  • Jurkat cell: 5 x 10^5 cell / well, add 22.5 pmol sgRNA and 2 μg-4 μg ssDNA/ 0.5 μg-3 μg dsDNA per well. (For HDR template lengths between 0.5-4 kb)
  • T cell: 1×10^6 cell / well, add 120 pmol sgRNA and 2 μg-6 μg ssDNA/ 2 μg dsDNA per well. (For HDR template lengths between 0.5-4 kb)
  • iPSC cell: 5 x 10^5 cell / well, add 7.5 pmol sgRNA and 2 μg-6 μg ssDNA/ 0.5 μg-3 μg dsDNA per well. (For HDR template lengths between 0.5-4 kb)

  We recommend a molar ratio of Cas9:sgRNA between 1:1 to 1:4.

  Here is the suitable reagent dosage used in GenScript R&D, which can be used for reference. Howwever, we highly recommand you optimize and find the suitable reagent dosage according to your specific experimental system.

• Besides sgRNA and an HDR template, what reagents will I need to perform a CRISPR-mediated knock-in?

  A Cas protein is required to recognize and cleave your target DNA sequence. The most commonly used Cas protein is SpCas9, which requires a NGG PAM sequence adjacent to the target site. However, there are other Cas variants that can recognize different PAM sequences or have increased specificity. You can either express the Cas protein from a plasmid or deliver it as a purified protein (ribonucleoprotein, RNP) into the cells. Depending on your desired method of delivery, you will also need a delivery system such as an electroporation system or lipid nanoparticle pakcaging, as well as necessary buffers and optional HDR knock-in enhancer.