CRISPR-Cas9 is revolutionizing genomic research by enabling efficient site-specific changes in the genomes of a wide variety of model organisms. After Cas9 generates a double-strand break (DSB), mammalian cells use endogenous DNA repair mechanisms to join the broken ends together. There are 2 main pathways that the cells follow to repair the break: non-homologous end joining (NHEJ) and homology-directed repair (HDR). As its name implies, the NHEJ pathway joins DSB ends without the need for a homologous template. However, NHEJ activity is error prone and introduces small insertions or deletions (indels) at the break point, potentially disrupting a target gene’s normal function. NHEJ is therefore useful for creating gene knockouts. Introducing precise, sequence-specific changes requires HDR, the second repair pathway. HDR requires a donor DNA fragment containing sequences identical to those flanking the break point, often referred to as homology arms, to enable homologous recombination by the endogenous cellular machinery [1]. Consequently, HDR can repair DSBs in an error-free manner. The donor DNA template can carry a change in sequence between the homology arms, or it can carry much larger fragments for insertion at or near the break point.
Advantages of ssDNA donor templates
Historically, double-stranded DNAs (dsDNA) have been used as the donor templates [2]. However, dsDNA donors are more readily incorporated by the dominant NHEJ process, resulting in duplication of homology arms or partial incorporation of the dsDNA template. Homology-independent insertion events by NHEJ can also occur at off-target DSBs or naturally occurring endogenous DSBs. In addition, dsDNA templates can be deleterious to cultured cells, and delivery of linear or plasmid dsDNAs often leads to poor transfection efficiency and adverse cellular responses [3, 4]. To avoid these undesired consequences, researchers turned their attention to single-stranded DNA (ssDNA) templates.
ssDNA donor templates, also known as single-stranded donor oligonucleotides (ssODNs), have long shown greater recombination efficiency in bacteria and lower eukaryotes [5]. Recently, ssDNAs carrying exogenous sequences have also shown superior performance in a variety of mammalian systems. ssDNAs allow shorter homology arms, while still providing higher insertion efficiencies compared to similar dsDNA templates [6, 7]. As a result, ssDNAs are becoming the preferred template for HDR applications.
As a global leader in manufacturing synthetic DNA, IDT now supplies Megamer Single-Stranded DNA Fragments. These long, high-fidelity sequences are well suited for CRISPR HDR experiments. Megamer Single-Stranded DNA Fragments are sequence-verified ssDNAs ranging from 200 to 2000 bases. These longer fragments enable complex site-specific edits, such as creation of conditional or insertional alleles in mice [8].
In addition to CRISPR-HDR, Megamer fragments can be used in applications such as in vitro transcription and assays that require long, high-fidelity ssDNA sequences.
Design considerations for donor templates
Careful optimization of experimental conditions and design of Megamer donor templates are critical for achieving high HDR rates. The position of the cut site, the length of the homology arms, the presence of SNPs or other sequence variants, as well as possible secondary structures and GC content are all important considerations for optimization.
- Ensure that the Cas9 enzyme cleavage site is close to the position of the desired change. The distance between the cut site and the position of the desired change affects HDR efficiency. We examined the importance of this distance in HEK-293 cells using short ssDNA templates. We observed a dramatic reduction of HDR rate when the template insertion is just 5–10 bases away from the cut site. We recommend that you keep the cleavage site in close proximity to the point of insertion. If the insertion site is fixed, hence limiting the guide choices, we recommend giving the priority to the activity of the guide over optimal distance, and avoiding low activity guides.
- Use 75–100 base homology arms. Homology arms are the sequences within a donor template that correspond to the genomic sequences immediately flanking the genomic cut site. The lengths of the homology arms in Megamer templates impact HDR efficiency. While robust HDR has consistently been observed with homology arms as short as 30–40 bases for short ssDNA templates, we recommend designing 75–100 base homology arms for the longer Megamer templates based on our research findings. This is due to the variation in HDR efficiency in different biological systems, as well as the possibility of exonuclease degradation.
- Avoid aligning homology arms with SNPs or other sequence variants. The presence of SNPs or other sequence variants will reduce HDR efficiency. We recommend that you verify the sequence in your specific cell lines (or model organisms) of interest because sequence variations may be present that are not characterized or annotated in reference genomes. Such variants can reduce the recombination efficiency.
- Avoid creating secondary structures and local GC content extremes. During design of Megamer fragments, unintended secondary structures or regions of high GC content may be created. Specifically, the terminal regions of a donor template, which are typically homology arms, are particularly sensitive to GC content. Based on understanding of oligo secondary structure formation from decades of experience in DNA synthesis, we recommend limiting the GC content of the 50 bases on each end of a Megamer fragment to within 28–72% to avoid secondary structures. If homology arms’ GC content does not fall within this range, we recommend shifting the homologous arms or lengthening them to meet this requirement.
- Consider these special design recommendations for Megamer fragments containing loxP sites.
The Cre-loxP system is a powerful, versatile system for intracellular DNA recombination. The system consists of the Cre recombinase enzyme and its 34 bp recognition sequence (loxP site). The orientation and location of the loxP sites determine how the genetic material flanked by these sites will be rearranged [8].
A loxP site consists of two 13 bp palindromic repeats separated by an 8 bp asymmetric core spacer element. The canonical loxP sequence contains the following sequence with the repeats shown in orange: ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. Multiple loxP variants exist with mutations in either the spacer or the repetitive sequence regions. Using these non-canonical loxP sites enables more efficient integration by reducing the chance of re-excision of the recombined product.
Because these long repeats allow for unwanted recombination, the insertion of two identical loxP sites into a sequence presents a manufacturing challenge. A tool embedded in the IDT ordering system automatically screens submitted Megamer sequences to identify these troublesome elements before an order is placed, and rejects those with 2 loxP sites too close together. If you have a donor template that absolutely requires tandem loxP sites, we recommend constructing a Megamer fragment of at least 800 bases in length and separating the 2 loxP sites by a minimum of 500 bases. Alternatively, you can use heterospecific loxP sites to decrease the complexity of the synthetic sequence [9].
Use our automated tool at www.idtdna.com/site/order/megamerentry to test sequences for complexity and identify any regions that are considered to be problematic for manufacturing.
Quality control of Megamer Single-Stranded DNA Fragments
IDT assembles Megamer fragments using the latest and most accurate manufacturing processes and, therefore, achieves near-clonal accuracy and the greatest purity available. We perform next-generation sequencing (NGS) verification and single-stranded capillary electrophoresis for each Megamer fragment to ensure that the delivered product is accurate and reliable. IDT pioneered the use of NGS-based Megamer sequence confirmation due to its demonstrated performance superiority over Sanger sequencing and ability to detect subpopulations that would otherwise go unnoticed by traditional QC methods. Megamer Single-Stranded DNA Fragments are typically shipped within 2–4 weeks of ordering.
