Question

When biologists use the CRISPR-Cas9 gene editing system, they rely on one of two types of gene repair to occur: non-homologous end joining (NEHJ) or homology directed repair (HDR). Explain the difference between these two types of repair, and why HDR in the presence of a “repair template” - desired sequence of external DNA, has the potential to allow for more precise gene editing.

Answer 1

Non-homologous end joining (NHEJ)

Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair. The term "non-homologous end joining" was coined in 1996 by Moore and Haber.

NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells.

NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells.^] In budding yeast (Saccharomyces cerevisiae), however, homologous recombination dominates when the organism is grown under common laboratory conditions.

When the NHEJ pathway is inactivated, double-strand breaks can be repaired by a more error-prone pathway called microhomology-mediated end joining (MMEJ). In this pathway, end resection reveals short microhomologies on either side of the break, which are then aligned to guide repair. This contrasts with classical NHEJ, which typically uses microhomologies already exposed in single-stranded overhangs on the DSB ends. Repair by MMEJ therefore leads to deletion of the DNA sequence between the microhomologies.

In bacteria

Many species of bacteria, including Escherichia coli, lack an end joining pathway and thus rely completely on homologous recombination to repair double-strand breaks. NHEJ proteins have been identified in a number of bacteria, however, including Bacillus subtilis, Mycobacterium tuberculosis, and Mycobacterium smegmatis. Bacteria utilize a remarkably compact version of NHEJ in which all of the required activities are contained in only two proteins: a Ku homodimer and the multifunctional ligase/polymerase/nuclease LigD. In mycobacteria, NHEJ is much more error prone than in yeast, with bases often added to and deleted from the ends of double-strand breaks during repair. Many of the bacteria that possess NHEJ proteins spend a significant portion of their life cycle in a stationary haploid phase, in which a template for recombination is not available. NHEJ may have evolved to help these organisms survive DSBs induced during desiccation. Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis, also encode Ku homologs and exploit the NHEJ pathway to recircularize their genomes during infection.^[12] Unlike homologous recombination, which has been studied extensively in bacteria, NHEJ was originally discovered in eukaryotes and was only identified in prokaryotes in the past decade.

Non-homologous end joining (NHEJ) and homologous recombination (HR) in mammals during DNA double-strand break

Homology directed repair (HDR)

Homology directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination. The HDR mechanism can only be used by the cell when there is a homologous piece of DNA present in the nucleus, mostly in G2 and S phase of the cell cycle. Other examples of homology-directed repair include single-strand annealing and breakage-induced replication. When the homologous DNA is absent, another process called non-homologous end joining (NHEJ) takes place instead.

Biological pathway

The pathway of HDR has not been totally elucidated yet (March 2008). However, a number of experimental results point to the validity of certain models. It is generally accepted that histone H2AX (noted as γH2AX) is phosphorylated within seconds after damage occurs. H2AX is phosphorylated throughout the area surrounding the damage, not only precisely at the break. Therefore, it has been suggested that γH2AX functions as an adhesive component for attracting proteins to the damaged location. Several research groups have suggested that the phosphorylation of H2AX is done by ATM and ATR in cooperation with MDC1. It has been suggested that before or while H2AX is involved with the repair pathway, the MRN complex (which consists of Mre11, Rad50 and NBS1) is attracted to the broken DNA ends and other MRN complexes to keep the broken ends together. This action by the MRN complex may prevent chromosomal breaks. At some later point the DNA ends are processed so that unnecessary residuals of chemical groups are removed and single strand overhangs are formed. Meanwhile, from the beginning, every piece of single stranded DNA is covered by the protein RPA (Replication Protein A). The function of RPA is likely to keep the single stranded DNA pieces stable until the complementary piece is resynthesized by a polymerase. After this, Rad51 replaces RPA and forms filaments on the DNA strand. Working together with BRCA2 (Breast Cancer Associated), Rad51 couples a complementary DNA piece which invades the broken DNA strand to form a template for the polymerase. The polymerase is held onto the DNA strand by PCNA (Proliferating Cell Nuclear Antigen). PCNA forms typical patterns in the nucleus of the cell through which the current cell cycle can be determined. The polymerase synthesizes the missing part of the broken strand. When the broken strand is rebuilt, both strands need to uncouple again. Multiple ways of "uncoupling" have been suggested, but evidence is not yet sufficient to choose between models (March 2008). After the strands are separated the process is done.
The co-localization of Rad51 with the damage indicates that HDR has been initiated instead of NHEJ. In contrast, the presence of a Ku complex (Ku70 and Ku80) indicates that NHEJ has been initiated instead of HDR.
HDR and NHEJ repair double strand breaks. Other mechanisms such as NER (Nucleotide Excision Repair), BER (Base Excision Repair) and MMR recognise lesions and replace them via single strand perturbation.

Mitosis

In the budding yeast Saccharomyces cerevisiae homology directed repair is primarily a response to spontaneous or induced damage that occurs during vegetative growth. In order for yeast cells to undergo homology directed repair there must be present in the same nucleus a second DNA molecule containing sequence homology with the region to be repaired. In a diploid cell in G1 phase of the cell cycle, such a molecule is present in the form of the homologous chromosome. However, in the G2 stage of the cell cycle (following DNA replication), a second homologous DNA molecule is also present: the sister chromatid. Evidence indicates that, due to the special nearby relationship they share, sister chromatids are not only preferred over distant homologous chromatids as substrates for recombinational repair, but have the capacity to repair more DNA damage than do homologs.^[5]

Meiosis

During meiosis up to one-third of all homology directed repair events occur between sister chromatids. The remaining two-thirds, or more, of homology directed repair occurs as a result of interaction between non-sister homologous chromatids.

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.

The CRISPR-Cas technology enables rapidand precise genome editing at any desired genomic positionin almost all cells and organisms. In this study, we analyzedthe impact of different repair templates on the frequency of ho-mology-directed repair (HDR) and non-homologous endjoining (NHEJ). We used a stable HEK293 cell line expressingthe traffic light reporter (TLR-3) system to quantify HDR andNHEJ events following transfection with Cas9, eight differentguide RNAs, and a 1,000 bp donor template generated either

The CRISPR-Cas technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology directed repair (HDR) and non-homologous end-joining (NHEJ). We used a stable HEK293 cell line expressing the Traffic Light Reporter system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1000 bp donor template generated either as circular plasmid, linearized plasmid with long 3’ or 5’ backbone overhang, or as PCR product. The sequence to be corrected was either centrally located , with a shorter 5’ homologous region), or a shorter 3’ homologous region . Guide RNAs targeting the transcriptionally active strand showed significantly higher NHEJ frequencies compared to guide RNAs targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5’ backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and thus, ultimately on the outcome of treatment approaches using HDR.

The evolution of genome editing technology based on CRISPR (clustered regularly interspaced short palindromic repeats) system has led to a paradigm shift in biological research. CRISPR/Cas9-guide RNA complexes enable rapid and efficient genome editing in mammalian cells. This system induces double-stranded DNA breaks at target sites and most DNA breakages induce mutations as small insertions or deletions by non-homologous end joining (NHEJ) repair pathway. However, for more precise correction as knock-in or replacement of DNA base pairs, using the homology-directed repair (HDR) pathway is essential. Until now, many trials have greatly enhanced knock-in or substitution efficiency by increasing HDR efficiency, or newly developed methods such as Base Editors (BEs). However, accuracy remains unsatisfactory. In this review, we summarize studies to overcome the limitations of HDR using the CRISPR system and discuss future direction.