1) CRISPR based gene insertion, replacement, or correction

Mechanism of CRISPR HDR based gene editing

CRISPR/Cas9 technology is commonly used to create precise double stranded breaks (DSBs) at target DNA sites. The guide RNA (gRNA) recognizes the protospacer adjacent motif (PAM) sequence on the target DNA after forming complex with Cas9, then Cas9 exerts its endonuclease function to cause DSBs. This triggers two mechanisms for repair: one is non-homologous end-joining (NHEJ), which introduces mutations in the DSB site. The other mechanism is homology directed repair (HDR) which enables the donor DNA to be inserted at the break site and create gene knock-ins.

Double-stranded DNA (dsDNA) was traditionally used as HDR donor DNA templates, however, recent studies demonstrated that single-stranded DNA (ssDNA or ssODN) is the best HDR templates for CRISPR based gene insertion, replacement, and correction^1-5. When compared to double-stranded DNA donors, ssDNAs demonstrated significantly improved editing efficiency and specificity, as well as reduced off-target integration, especially in editing primary cells, stem cells, and developing transgenic animal model.

2) In vitro transcription

In vitro transcription (IVT) method describes the transcription of RNA molecules via RNA phage polymerase (T7, T3 or SP6) using DNA templates in vitro. Three different types of DNA templates can be used for IVT, including circular plasmid DNA, (PCR-amplified) DNA fragments, and single-stranded DNA (ssDNA or ssODN). Resulting RNA transcripts are used in various applications, including analytical procedures, chemical studies, genetic studies, structure studies. In addition, artificial functional nucleic acids, such as aptamers and ribozymes, can also be produced through IVT.

Case Studies:

• High gene knock-in efficiency and reduced off target integration with single-stranded DNA HDR template
• Using Long ssDNA for the Generation of Foxi1-rtTA Transgenic Mice

User Manual:

• ssDNA User Manual

Webinars:

• CRISPR in Creating Knockin Cell Lines and Animal Models - Functionalizing Genome Editing for a Broad Range of Targets
• In vivo delivery of Cas9 ribonucleoprotein and donor DNA with gold nanoparticles
• CRISPR Based T Cell Editing: Large knock-ins in human T cells using non-viral HDR templates
• Strategies to Efficiently Generate CRISPR KO/KI Cell Lines

Related Publications:

When compared to double-stranded DNA donors, ssDNAs demonstrated significantly improved editing efficiency and specificity, as well as reduced off-target integration, especially in editing primary cells, stem cells, and developing transgenic animal model^1-5.

• "We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement." Miura H, et al., Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. (2018) 13(1):195-215.
• "We report that targeted chromosomal translocations are generated more efficiently when the all-in-one plasmid, RNP complex, and ssODN-based approaches are used, with the most efficient strategy being the combination of RNP complexes with translocation-ssODNs." Torres-Ruiz, et al., Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9. Stem Cell Reports. (2017) 8: 1408–1420.
• "ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration." Li, et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv 178905; doi: https://doi.org/10.1101/178905.
• "The PITCh approach required 265 zygotes whereas Easi-CRISPR used only 105 zygotes, and the PITCh approach produced 33% correctly targeted pups, whereas Easi-CRISPR (using ssDNA donors) produced 100% correctly targeted pups." Quadros, et al., Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biology (2017) 18:92.
• "Similar to what has been described with viral HDR templates, we found evidence to suggest that double-stranded templates could integrate independent of target homology, albeit at low rates. These rare events could be reduced almost completely by using single-stranded DNA (ssDNA) templates." Roth, et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559 (2018) 405–409.

• 1) How to choose from various HDR templates when performing CRISPR mediated gene KI? Should I choose dsDNA, plasmid DNA, or ssDNA? Read More »

It depends on the experimental purpose and host cell line. When compared to double-stranded DNA donors, ssDNAs demonstrated significantly improved editing efficiency and specificity, as well as reduced off-target integration, especially in editing primary cells, stem cells, and developing transgenic animal model.

	Plasmid DNA	dsDNA	ssDNA
HDR efficiency	Medium	High	Comparable to dsDNA
Off-target rate	Medium	High	Low
Cost	Lower	Medium	High
Toxicity	High	High	Low

• 2) What are the tips for designing an effective ssDNA template? Read More »

For point mutation, it is suggested to use asymmetric ssDNA design. You can read this paper for more design tips. For large gene insertion, homology arms with 300-1000 bp flanking the insertion gene have been reported. In most cases, 500 bp long homology arms should work. It is important to design the ssDNA template containing a silent mutation to mutate the sgRNA PAM sequence in order to avoid secondary cleavage. KI donor design can be complicated. It is highly suggested to use a software (e.g. snapgene) to view and edit the sequence.

• 3) What is the recommended homology arm length on each side of the template DNA when designing ssDNA? Read More »

Homology arms with 300-1000 bp flanking the insertion gene have been reported. In most cases, 500 bp long homology arms should work.

• 4) Any tips for improving CRISPR KI efficiency? Read More »

• Carefully design your sgRNA. The sgRNA cleavage site should be close to the mutation site and it is best to be within 15 bp of each other.
• Optimize the transfection efficiency before transfection of gRNA/Cas9 and donor template.
• Use FACS sorter to enrich the transfected cells.
• Use drug or marker to select KI clones if feasible (e.g tag a puromycin resistance gene at C-termini of insertion gene for selection).
• For more information, please view GenScript's webinar on Strategies to Efficiently Generate CRISPR KO/KI Cell Lines.

• 5) Any tips for generation of conditional alleles in mouse? Read More »

CRISPR/Cas9-mediated null allele production in mice is highly efficient, however, the generation of conditional alleles has proven to be more difficult.

• HDR efficiency is highly correlated with Cas9 cutting efficiency, thus can be improved by carefully designing and choosing sgRNAs.
• Use long ssDNA, rather than dsDNA, for significantly reduced off-target integration.
• While generating conditional alleles via the integration of LoxP sequence, use one long ssDNA is more efficient than using two short ssDNAs.

• 6) What if low HDR efficiency is detected in transfected cells? Read More »

You can refer to the following workflow to identify issues and solutions:



• 7) What is the maximum ssDNA length GenScript produces? Read More »

At GenScript, sequences that are less than 3000 nt long in length are routinely produced. We have also successfully synthesized ssDNA that are up to 5000 nt long with motifs of extremely high GC content and repeat regions in the past. If you want to synthesize ssDNAs longer than 3000 nt long, please contact our Technical Support Team to get a quote.

• 8) What is the maximum quantity GenScript provides? Read More »

At GenScript, the maximum delivery quantity for sequences < 1 kb long is 40 µg; for sequences within 1-3 kb long is 20 µg; and for sequences >3 kb long, the maximum yield is 5 µg.

• 9) What are the quality control tests you provide for the ssDNA templates? Read More »

We carry out two major QC examinations on the final ssDNA product prior to lyophilization: 1) Sanger sequencing to ensure the accuracy of the ssDNA sequence; 2) Purity test of the final ssDNA product via gel electrophoresis.

• 10) Why do you do double-sequencing, including the final ssDNA product? Read More »

During the production of ssDNA, we do two rounds of sequencing to guarantee the sequence accuracy. We first pick sequence verified plasmid DNA template via sequencing to ensure the purity and sequence accuracy of the final ssDNA product. In addition, we use direct sequencing on the final ssDNA product to confirm the ssDNA product homogeneity.

• 11) How do you purify the final ssDNA product? Read More »

We combine the advantages of using magnetic beads and agarose gel purification to ensure high yield, absolute purity, and zero chemical contaminations.

• 12) Do I need to worry about dsDNA contamination in the ssDNA product? Read More »

No. The final ssDNA product only contains full length, sequence-verified, ssDNA molecules. By using our proprietary, patent-pending, enzymatic synthesis approach, GenScript guarantees that our ssDNA product has a non-detectable dsDNA level.

• 13) Why is there more than one band in the gel image on my COA report? Read More »

Some ssDNA products may have strong aggregation tendency due to the nature of their nucleotide sequences. These aggregates are most likely formed due to intramolecular and/or intermolecular forces.

To test whether the extra band in a gel image is ssDNA aggregates or dsDNA contamination, you can perform a digestion test using S1 Nuclease, which degrades ssDNA, but not dsDNA. If the ssDNA product is 100% pure, with no dsDNA, then all samples should be digested with S1 Nuclease and no band should be observed after running gel electrophoresis. However, if a product has dsDNA contamination, then it can't be fully digested after the addition of S1 Nuclease and still have bands present on the gel image.



To further confirm whether the extra band is indeed ssDNA aggregates, you can cut out the ssDNA band on the gel and run a second around of gel electrophoresis. If the extra band is aggregated ssDNA molecules, it will still show up on the second gel after purification. In addition, the ratio of the extra band over the ssDNA band in the two gels would remain similar.