This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill.
One advantage to using the CRISPR/Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses. After the initial cut, the next steps in the process involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways – Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could be relevant when planning your experiment. This blog has previously considered the HDR pathway; below we’ll discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome.
Non-Homologous End Joining
Unlike HDR, NHEJ is active throughout the cell cycle and has a higher capacity for repair, as there is no requirement for a repair template (sister chromatid or homologue) or extensive DNA synthesis. NHEJ also finishes repair of most types of breaks in tens of minutes – an order of magnitude faster than HDR. NHEJ is consequently the principle means by which CRISPR/Cas9-introduced breaks are repaired.
The following factors are required for NHEJ repair regardless of end structure, and dictate the major events of the pathway:
- Broken ends are recognized by loading of the Ku70/Ku80 heterodimer
- Ku then acts as a scaffold for recruitment of a kinase (DNA-PKcs) and a two subunit DNA ligase (XRCC4-ligase IV); together with some accessory factors (PAXX, XLF), this complex holds a pair of DNA ends together, forming a paired end complex
- The paired end complex then ligates compatible DNA ends together, thus repairing the break.
This is a simplified, streamlined version of this pathway and does not consider the missing or damaged nucleotides that are common to biological sources of DSBs, and which need to be processed. Processing occurs prior to ligation as incompatible DNA ends interfere with that step. Accordingly, NHEJ has a vast toolbox of processing factors, including polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific end cleaning enzymes (Aprataxin, Tdp2) that function to make ends better substrates for ligation. Although we do not describe these steps here, the processing of DNA ends tends to be the point where mutations are introduced.
Repair of Cas9-induced breaks by NHEJ
As illustrated below, NHEJ-mediated repair of Cas9-generated breaks is useful if the intent is to make a null allele (“knockout”) in your gene of interest, as it is prone to generating indel errors. Indel errors generated in the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently about a two-thirds chance of causing a frameshift mutation. Of some importance, the deletion can be less heterogeneous when constrained by sequence identities in flanking sequence (“microhomologies”).
It must be emphasized that NHEJ doesn’t obligatorily introduce indels; given the end structure of the Cas9 DSB (blunt or near-blunt ends without nucleotide damage) such products are rare, probably accounting for less than 5% of repair events. However, the products of accurate repair are easily re-cleaved while indel products aren’t, so repeated cycles will favor accumulation of the latter products. As noted above, a single cycle of cleavage and accurate repair should take less than an hour, thus a population of cells constitutively expressing a targeted Cas9 should possess indels in the majority of their chromosomes within a day. Another factor expected to impact repair is that the Cas9 protein doesn’t immediately release from the broken end after cleavage, which may interfere with loading of Ku and normal NHEJ activity.
NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that flank regions of hundreds of base pairs or more can simultaneously introduce a pair of chromosome breaks, and could result in deletion of the intervening DNA (“pop-out” deletions) if NHEJ joins the distal ends together. Similarly, it may be possible to direct insertion of an exogenous DNA fragment at a Cas9 targeted break (or pair of breaks) by NHEJ-dependent repair (“pop-in” insertion) provided a template containing compatible overhangs is available. Cas9 can also be altered to generate a targeted single strand break; when two such breaks are introduced near each other, in opposite strands, it’s presumed that this results in a DSB with long overhangs. This “double nickase” strategy vastly reduces breaks and mutations at off-target sites, but it is not yet clear how NHEJ engages this class of breaks.
Thank you to our guest bloggers!
David Wyatt is a graduate student interested in determining how the structure of broken ends impacts how they are repaired. He works in Dale’s lab.
Dale Ramsden is a member of the curriculum in Genetics and Molecular Biology, the Dept. of Biochemistry and Biophysics and the Lineberger Comprehensive Cancer Center at UNC Chapel Hill.
Useful links and References
Addgene's CRISPR guide: http://www.addgene.org/crispr/guide/
Find CRISPR plasmids: http://www.addgene.org/crispr/
General NHEJ References:
Limiting the persistence of a chromosome break diminishes its mutagenic potential. Bennardo, N., Gunn, A., Cheng, A., Hasty, P., & Stark, J. M. PLoS Genet, 5(10), e1000683. (2009). PubMed.
Is non-homologous end-joining really an inherently error-prone process? Bétermier, M., Bertrand, P., & Lopez, B. S. PLoS Genet, 10(1), e1004086. (2014). PubMed.
The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. Waters, C. A., Strande, N. T., Pryor, J. M., Strom, C. N., Mieczkowski, P., Burkhalter, M. D., et al. Nature Communications, 5, 4286. (2014). PubMed.
Chromosomal translocations in human cells are generated by canonical nonhomologous end- joining. Ghezraoui, H., Piganeau, M., Renouf, B., Renaud, J.-B., Sallmyr, A., Ruis, B., et al. Molecular Cell, 55(6), 829–842. (2014). PubMed.
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., et al. Cell, 154(6), 1380–1389. (2013). PubMed.
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