CRISPR 101: Homology Directed Repair

Originally published Mar 12, 2015 and last updated Jul 27, 2020.

DNA lesions are sites of structural or base-pairing damage of DNA. Perhaps the most harmful 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.

How does homology directed repair fix DNA double-strand breaks? 

Genome stability requires 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 is the canonical homology-independent pathway as it involves the alignment of only one to a 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 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  specific mutations into the damaged DNA.

There are four different HDR pathways to repair DSBs. Here are  three central steps of the HDR pathways:

1. 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.
   
   
2. 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). This is the defining point of HDR.
   
   
3. The recombination intermediates can then be resolved to complete the DNA repair process

Homology directed repair pathways

HDR can occur either non-conservatively or conservatively. The non-conservative method is composed of the single-strand annealing (SSA) pathway and is more error prone.. The conservative methods, characterized by the accurate repair of the DSB by means of a homologous donor (e.g., sister chromatid, plasmid, etc), are 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 (DSBR)

In the classical DSBR pathway, the 3’ ends invade an intact homologous template to 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. 

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. In general, the insertion sites of the modification should be very close to the DSB, ideally less than 10 bp away if possible. Check out our blog post on Improving HDR efficiency for more details on how "Cut-to-Mutation distance" affects editing efficiency.

One important point to note is that the CRISPR enzymes may continue to cleave DNA 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 repairing the DNA. This repeated editing is problematic if you are trying to introduce a very specific mutation or sequence. To get around this, you should design the HDR template in such a way that will ultimately block further Cas9 targeting after the initial DSB is repaired. Two common ways to block further editing are mutating the PAM sequence or the gRNA seed sequence.

What makes the best repair template: plasmid DNA or single-stranded donor oligonucleotide (ssodn)?

When designing your repair template, the size of your intended edit is a big factor. ssDNA templates (referred to as ssODNs) are commonly used for smaller modifications. Small insertions/edits may require as little as 30-50 bases for each homology arm, but keep in mind these numbers may vary based on your locus of interest and experimental system. 50-80 base homology arms are commonly used. Asymmetric homology arms (36 bases distal to the PAM and 91 bases proximal to the PAM) supported HDR efficiencies up to 60% (Corn et al., 2016).

Due to the difficulties associated with creating ssODNs longer than 200 bases, researchers have used dsDNA plasmid templates for larger insertions such as fluorescent proteins or selection cassettes. These templates should have homology arms of at least 800 bp. HDR efficiency with plasmid templates is generally low; to increase the frequency of edits, researchers have designed self-cleaving plasmids that contain gRNA target sites flanking the template. When the Cas enzyme and the appropriate gRNA(s) are present, the template is liberated from the vector. To avoid plasmid cloning, scientists also use PCR-generated long dsDNA templates, but these templates may be toxic to cells, thus lowering editing efficiency. Another disadvantage of dsDNA templates is their ability to bluntly integrate into the genome, duplicating the homology arm sequences. 

Easi-CRISPR, a technique to allow researchers making large mutations to take advantage of the benefits of ssODNs (Quadros et al., 2017). To create ssODNs longer than 200 bases, you in vitro transcribe RNA encoding your repair template, then use reverse transcriptase to create the complementary ssDNA. Easi-CRISPR works well in mouse knock-in models, increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs. Although HDR efficiency varies across loci and experimental systems, ssODN templates generally provide the highest frequency of HDR edits.  Check out our blog post on Easi-CRISPR for more details.

Promoting HDR over NHEJ

To promote HDR over the more common NHEJ repair mechanism, strategies such as inhibiting NHEJ and activating HDR have been used. Rees et al. and Nambiar et al. have used HDR factors such as Rad variants like Rad18 (Nambiar et al., 2019) and hRad51 (Rees et al., 2019) to activate HDR. Inhibitors such as CYREN can be used to inhibit NHEJ (Arnoult et al., 2017), while a Cas9 fusion to dominant negative 53BP1 both inhibits NHEJ and activates HDR (Jayavaradhan et al., 2019).

Useful links and references:

Want to know more? Check out the additional resources below for more in-depth information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles driving CRISPRs and genome editing technology.

Links:

• Zhang Lab’s CRISPR FAQs
• CRISPR forum moderated by the Zhang lab
• CRISPR Challenges: Standardization and HDR
• CrispyCrunch
• Optimizing Donor DNA for Enhanced CRISPR Genome Editing

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.
  
• The Road Less Traveled: Strategies to Enhance the Frequency of Homology-directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis (2018). Pubmed.

CRISPR References:

• 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.

• Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Richardson CD, et al. Nat Biotech. 34(3); (2016). PubMed.

• Efficient homology-directed repair using single-stranded DNA templates. Jacobi SA, et al. Poster at Keystone Precision Genome Engineering. 2017. 

• Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.