DNA lesions are defined as sites of structural or base-pairing damage of DNA. Perhaps the most nocuous type of lesion results from breakage of both DNA strands – a double-strand break (DSB) – as repair of DSBs is paramount for genome stability. DSBs can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces such as ionizing radiation and ultraviolet light; however, these types of breaks occur randomly and unpredictably. To provide some control over the location of the DNA break, scientists have engineered plasmid-based systems that can target and cut DNA at specified sites. Regardless of what causes the DSB, the repair mechanisms function in the same way.
In this post, we will describe the general mechanism of homology directed repair with a focus on repairing breaks engineered in the lab for genome modification purposes.
Mechanisms to Repair DNA Double-Strand BreaksGenome stability necessitates the correct and efficient repair of DSBs. In eukaryotic cells, mechanistic repair of DSBs occurs primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). NHEJ (discussed in a future post) is the canonical homology-independent pathway as it involves the alignment of only one to few complementary bases at most for the re-ligation of two ends, whereas HDR uses longer stretches of sequence homology to repair DNA lesions. This post focuses on HDR, which is considered to be the more accurate mechanism for DSB repair due to the requirement of higher sequence homology between the damaged and intact donor strands of DNA. The process is error-free if the DNA template used for repair is identical to the original DNA sequence at the DSB, or it can introduce very specific mutations into the damaged DNA.
The three central steps of the HDR pathways are listed as follows:
- The 5’-ended DNA strand is resected at the break to create a 3’ overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis.
- The invasive strand can then displace one strand of the homologous DNA duplex and pair with the other; this results in the formation of the hybrid DNA referred to as the displacement loop (D loop).
- The recombination intermediates can then be resolved to complete the DNA repair process.
The invasion of the 3’ single-stranded DNA (ssDNA) into the homologous DNA duplex (Step 2) is the defining point of HDR. There are four different HDR pathways that can be employed to repair DSBs and the specific mechanisms used in Steps 2 and 3 define the individual pathways as described below.
Double-Strand Break Repair Pathways Associated with HDR
HDR can occur either non-conservatively or conservatively. The non-conservative method is composed of the single-strand annealing (SSA) pathway and, in the interest of space, will not be discussed here. The conservative methods, characterized by the accurate repair of the DSB by means of a homologous donor (e.g., sister chromatid, plasmid, etc), is composed of three pathways: the classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced repair (BIR).
Classical Double-Strand Break Repair
In the classical DSBR pathway, the 3’ ends invade an intact homologous template then serve as a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (dHJs). dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and synthesizes DNA from the second DSB end. The individual HJs are resolved via cleavage in one of two ways. Looking at the left branch of the figure below, each junction resolution could happen on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at the orange arrows). If resolved dissimilarly (e.g. one junction is resolved on the crossing strand and the other on the non-crossing strand), a crossover event will occur; however, if both HJs are resolved in the same manner, this results in a non-crossover event. DSBR is semi-conservative, as crossover events are most common. This animation nicely illustrates the DSBR pathway: http://web.mit.edu/engelward-lab/animations/DSBR.html.
Synthesis-Dependent Strand-Annealing Pathway
As illustrated on the right branch of the figure above, SDSA is conservative, and results exclusively in noncrossover events. This means all newly synthesized sequences are present on the same molecule. Unlike DSBR, following strand invasion and D loop formation in SDSA, the newly synthesized portion of the invasive strand is displaced from the template and returned to the processed end of the non-invading strand at the other DSB end. The 3’ end of the non-invasive strand is elongated and ligated to fill the gap, thus completing SDSA.
Break-Induced Repair Pathway
Although BIR is not as well characterized as either DSBR or SDSA, one central feature of this pathway is the presence of only one invasive end at a DSB that can be used for repair. This single invasive strand invades a homologous sequence and initiates both leading and lagging strand synthesis, which results in the formation of one HJ. This HJ is resolved by cleavage of the crossed strand. While this pathway may not be immediately applicable in DSB-induced gene targeting or relevant to plasmid-based genome engineering, it may have biological importance for the repair of chromosome ends that have no second end that would enable DSBR or SDSA.
Repair of DSBs and Genome Engineering
The advent of plasmid-based methods to induce DSBs engendered numerous technologies that integrated homologous recombination into genome engineering efforts. Early meganuclease-based technologies established the foundation for plasmid-based genome engineering tools such as Zinc Finger Nucleases (ZFNs) and TAL Effector Nucleases (TALENs). ZFNs and TALENs can both be used to direct an endonuclease to a specific DNA locus targeted for modification. On the surface, the discovery and development of RNA-guided CRISPR/Cas9 technology may just appear as a new method for directing a nuclease to cause a specific DSB; however, the ease of creating guides, the speed of the system, and the overall versatility in application has not just reinvigorated genome engineering, but has really revolutionized the field.
General Considerations for Designing a Repair Template to Create Mutations
HDR templates used to create specific mutations or insert new elements into a gene require a certain amount of homology surrounding the target sequence that will be modified. Scientists have been most successful using homology arms that start at the CRISPR-induced DSB; however, there may be some wiggle room. In general, the insertion sites of the modification should be no more than 100bp away from the DSB, ideally less than 10bp away if possible, and the overall length of the homology arm is an important factor to consider when designing these (more on this below). Longer distances of up to 200bp may work, but the efficiency will likely be lower and you may need to introduce a selection marker to ensure the modification is present.
One important point to note is that the CRISPR/Cas9 system does not stop once a DSB is introduced and repaired. As long as the gRNA target site/ PAM site remain intact, the Cas9 endonuclease will keep cutting and the DSB will keep getting repaired through either NHEJ or HDR. This could be problematic if you are trying to introduce a very specific mutation or sequence. To get around this, one may consider designing the HDR template in such a way that will ultimately block further Cas9 targeting after the initial DSB is repaired.
What Makes the Best Template: Plasmid DNA or Single-stranded Donor Oligonucleotide (ssODN)?
The size of your intended mutation is a big factor in deciding on a single- or double-stranded DNA repair template. Historically plasmids have been used as dsDNA templates when creating gene-targeting vectors; however, ssDNA templates (ssODNs) have come into common use for smaller modifications as they tend to have a higher efficiency. As a basic guideline, small mutations of up to ~50bp or single point mutations can successfully be introduced using ssODN templates, while dsDNA plasmid-based templates should be used for larger inserts such as fluorescent proteins or selection cassettes.
For ssODNs, the templates should be as long as possible with the Cas9-induced break point centered within the template. Scientists have been successful with template lengths of ~100-200bp in total, with at least 40bp (but usually closer to 50-80bp) homology arms on either side of your intended mutation. Because target sequence placement is PAM-dependent, it is not always possible to have the insertion site right next to the cut site; however, they should be reasonably close (within ~20bp) to each other.
For larger inserts, dsDNA encompassing homology arms of 800bp each or larger should be used. Plasmids are the most common source for providing dsDNA targets; however, they will need to be linearized before transfection. This webpage is a great resource for designing a gene targeting vector: http://ko.cwru.edu/info/targvectdesign.html.
Useful Links and References:
Want to know more? Check out the additional resources below for more in-depth information on HDR and CRISPRs. Watch for new CRISPR 101 posts as we continue this series to help explain the basic principles driving CRISPRs and genome editing technology.Links:
- CRISPR forum moderated by the Zhang lab: https://groups.google.com/forum/#!searchin/crispr/HDR
- CRISPR plasmids: http://www.addgene.org/crispr/
General Homologous Recombination References:
- Repair of Strand Breaks by Homologous Recombination. Jasin M and Rothstein R. Cold Spring Harb Perspect Biol (2013). PubMed.
- DNA double-strand break repair: how to fix a broken relationship. Pardo B, et al. Cell Mol Life Sci (2009). PubMed.
- Regulation of DNA double-strand break repair pathway choice. Shrivastav M, et al Cell Research (2008). PubMed.
- Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Pfeiffer P, et al. Mutagenesis (2000). PubMed.
- Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Paques F and Haber JE. Microbiology and Molecular Biology Reviews (1999). PubMed.
- Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013). PubMed.
- A Mouse Geneticist's practical guide to CRISPR applications. Singh P, et al. Genetics 114.169771; (2014). PubMed.
- The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations. Edited by Doudna J and Sontheimer EJ. Methods in Enzymology Vol. 546; (2015). PubMed.
- Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System. Li, K et al. PLoS ONE 9(8); (2014). PubMed.
- Making designer mutants in model organisms. Peng, Y. et al. Development 141; (2014). PubMed.
Note: Marcy Patrick contributed to the writing of this article.
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