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Alt-R™ HDR Donor Blocks

Built for homology-directed repair experiments

For researchers looking to accelerate their CRISPR, HDR-mediated insertions (>120 bases), IDT dsDNA templates offer a cost effective, high-fidelity option to reduce the amount of downstream screening via higher HDR rates and lower unintended integrations

  • Ideal for making longer than 120 bp genomic insertions
  • Sequence-verified via next-generation sequencing
  • Modified to increase successful HDR events
  • Lower unintended and non-homologous (blunt) integrations

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Alt-R HDR Donor Blocks

HDR Donor Blocks include chemical modifications within universal, non-integrating terminal sequences to help reduce unwanted blunt integration events.

Alt-R HDR Design Tool

If you don’t have a template design of your own, use our Alt-R HDR Design Tool to design your template. Simply provide basic information about your target site, and then use the tool to design and visualize your desired edit within the sequence. The Alt-R HDR Design Tool will provide the recommended HDR donor template along with gRNA(s) for your specifications.

Alt-R HDR Donor Blocks were developed to address the need for better HDR research solutions when creating larger changes or inserts in the genome. Utilizing the same high-fidelity process as IDT gBlocks™ HiFi Gene Fragments, the HDR Donor Blocks incorporate advanced chemical modifications at each end of the sequence to boost HDR rates and aid in inhibiting the occurrence of blunt-end integration of the donor sequence. In addition, universal sequences are added to the ends of the sequence ordered to provide consistency and speed of production.

Alt-R HDR Donor Blocks are available from 201 to 3000 bases in length and are generated from clonally purified DNA. The donors are sequence-verified via next generation sequencing and are typically shipped within 17–20 business days*. They are composed of A, T, G, and C nucleotides only. Sequence information is always secure and confidential at IDT. Non-disclosure agreements are available through IDT legal services upon request.

* The time required to manufacture an Alt-R HDR Donor Block is dependent on many factors and in a few cases may exceed the estimated delivery time.

Alt-R™ HDR Donor Blocks offer an improved solution for efficient generation of large knock-ins compared to long, single-stranded DNA templates

We investigated the HDR efficiency of inserting a green fluorescent protein (GFP) tag using either long, single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) templates (700 bp insert, 200 bp homology arms). The combined use of the modified Alt-R HDR Donor Blocks and the Alt-R HDR Enhancer V2 gave the higher HDR rates compared to no treatment when tested at multiple genomic loci and in multiple cell lines (Figure 1).

Figure 1. Alt-R HDR Donor Blocks offer improved large knock-in rates relative to long ssDNA HDR templates.

HEK-293 and K562 cells were electroporated with 2 µM Cas9 RNP complexes targeting three genomic loci along with 50 nM dsDNA or ssDNA donor templates using the Nucleofector™ system (Lonza). HDR templates were designed to mediate a GFP-tagging event (700 bp insert, 200 bp homology arms). The dsDNA templates contained either no modifications (unmodified), or the Alt-R HDR Donor Block modification. Both the targeting (T) and non-targeting (NT) strands were tested for the long ssDNA templates. After electroporation, cells were plated in media with or without 1 µM Alt-R HDR Enhancer V2 with a media change after 24 hours. Genomic DNA was isolated 48 hours after electroporation. Editing was assessed by long-read amplicon sequencing on the MinION™ system (Oxford Nanopore Technologies) using R9.4.1 chemistry and Guppy High Accuracy (HAC) basecalling. Reads were mapped to the unedited reference amplicon or desired HDR outcome using minimap2 (-x map-ont). A read was classified to be derived from the HDR pathway if it preferentially mapped to the desired HDR outcome. HDR efficiency was measured by calculating the read counts classified as HDR relative to the total aligned reads per sample. Error bars represent the standard error of two biological replicates per cell line.

Combined use of Alt-R HDR Donor Blocks and Alt-R HDR Enhancer V2 assist in mitigating the risk for off-target integration events

The use of unmodified dsDNA templates poses a risk for unwanted off-target integrations, where the donor is directly ligated into either a double-strand break (DSB) resulting from a Cas9 off-target editing event, or a naturally occurring endogenous DSB. We explored the ability of the Alt-R HDR Donor Block modification to reduce the occurrence of this unwanted repair event by generating a mock off-target DSB. Unmodified or Alt-R modified dsDNA templates for four unique HDR events were co-delivered with a Cas9 RNP targeting a site lacking homology to the donor templates. The combined use of Alt-R HDR Donor Blocks and the Alt-R HDR Enhancer V2 substantially reduced the rates of integration at a non-homologous DSB, as compared to no treatment (Figure 2).

Total editing at the mock off-target site was >90% (data not shown). As such, the reported data represent much higher integration frequencies than would be expected at true Cas9 off-targets, or at endogenous DSBs where overall editing frequencies would be lower. Any strategies that reduce the risk for Cas9 off-target editing (such as use of Alt-R HiFi Cas9 Nuclease) further mitigate the risk for off-target integration of the HDR template.

Figure 2. Combined use of Alt-R HDR Donor Blocks and Alt-R HDR Enhancer V2 reduces the rate of non-homologous (blunt) integrations at off-target DSBs.

A mock off-target DSB was generated by delivering 2 µM Cas9 RNP targeting the SERPINC1 locus into HEK-293 cells using the Nucleofector™ system (Lonza). The dsDNA donor templates mediating GFP insertions at alternative genomic loci (50 nM dose, n=4 sequences) were co-delivered with the mock off-target RNP. After electroporation, cells were plated in media with or without 1 µM Alt R HDR Enhancer V2 with a media change after 24 hours. Genomic DNA was isolated 48 hours after electroporation. The SERPINC1 locus was PCR-amplified and blunt insertion events were measured by size discrimination on a Fragment Analyzer (Agilent).

Use of optimal homology arm lengths improved HDR efficiency

We systematically varied the homology arm length for Alt-R HDR Donor Blocks mediating insertions ranging from 120 to 2000 bp at different genomic loci. Overall, HDR efficiency was improved when using homology arm lengths of 200–300 bp (Figure 3). While HDR rates varied with site and insertion size, use of Alt-R HDR Donor Blocks and Alt-R HDR Enhancer V2 led to efficient knock-in of sequences up to 2000 bp. Additional design recommendations for HDR templates can be found in the application note “Optimized methods for CRISPR-Cas9 homology-directed repair (HDR) for efficient, high-fidelity genome editing.

Figure 3. Homology arm lengths of 200–300 bp result in the highest HDR efficiency when using Alt-R HDR Donor Blocks.

K562 cells were electroporated with 2 µM Cas9 RNP complexes targeting four genomic loci and Alt-R HDR Donor Blocks mediating 120, 500, 700, or 2000 bp insertions using the Nucleofector™ system (Lonza). Donor templates were designed with homology arm lengths varying from 40 bp up to 500 bp, and were delivered at equal molar or equal mass amounts (100 nM or 1.2 µg for 120 bp insert, 50 nM or 0.9 µg for 500 bp insert, 50 nM or 1.5 µg for 700 bp insert, and 25 nM or 1.3 µg insert for 2 kb insert). After electroporation, cells were plated in media with or without 1 µM Alt-R HDR Enhancer V2 with a media change after 24 hours. Genomic DNA was isolated 48 hours after electroporation. Editing was assessed by long-read amplicon sequencing on the MinION™ system (Oxford Nanopore Technologies) as previously described (Figure 1). Error bars represent the standard deviation of two biological replicates.

Sequence Information is secure and confidential at IDT. Please see our Confidentiality Statement for more information. All online ordering steps, including sequence entry and your choice of parameters, are also secure and protected.

We screen the sequence of every gene fragment order we receive to (1) identify any regulated and other potentially dangerous pathogen sequences, and (2) verify that IDT’s gene customers are legitimate scientists engaged in beneficial research.

IDT is among the five founding members of the International Gene Synthesis Consortium (IGSC) and helped to create the IGSC’s Harmonized Screening Protocol. The Harmonized Screening Protocol describes the gene sequence and customer screening practices that IGSC member companies employ to prevent the misuse of synthetic genes. IDT takes the steps set out in the Harmonized Screening Protocol to screen the sequences of ordered genes and the prospective customers who submit those orders.

For more information about the IGSC and the Harmonized Screening Protocol, please visit the website at genesynthesisconsortium.org.

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In October 2010, the United States government issued Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic genes should perform gene sequence and customer screening. IDT and the other IGSC member companies supported the adoption of the Screening Framework Guidance, and IDT follows that Guidance in its application of the Harmonized Screening Protocol.

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*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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