Basics of genome structure
Most of the DNA in eukaryotes, i.e. animals, plants and fungi, is present in stranded chromosomes. These consist of chromatin, which is DNA wrapped many times around histone proteins, and other DNA-binding proteins. In chromatin, DNA is shortened many times, very compactly organized and also protected. The structure of the chromatin can vary section by section, for example, in that the DNA is methylated to different degrees or the histone proteins are acetylated (epigenetic changes). Both alter the chromatin structure and determine whether the DNA is accessible: If the DNA is loosely packed, the genes it contains can be replicated and expressed by enzymes. The loose form is called euchromatin. In contrast, the DNA in heterochromatin is densely packed and largely inactive.
Results of the Weiss et al. study
The current study investigated whether the chromatin structure has an influence on the effectiveness of the CRISPR/Cas9 gene scissors and how the genome is changed in the process. For this purpose, the researchers screened the genome of the model plant Arabidopsis thaliana for identical CRISPR/Cas9 target sequences present in the different chromatin structures which were, therefore, suitable for comparison. CRISPR/Cas9 was then stably integrated into the genome of the plant cells and the outcome of the genetic scissor application was checked using next generation sequencing, i.e. state-of-the-art DNA sequencing.
It was found that CRISPR/Cas9 altered the DNA in heterochromatin regions less frequently than in euchromatin regions. Nevertheless, CRISPR/Cas9 effectiveness also varied differently in the euchromatin regions studied.
To increase CRISPR/Cas9 effectiveness in heterochromatin, the number of DNA methylations associated with heterochromatin was reduced. For this purpose, an enzyme that methylates DNA was turned off and the genome was treated with a chemical that reduces DNA methylations. This did, in fact, increase the frequency of mutagenesis, i.e. the number of changes in some areas of the heterochromatin.
Further analysis should answer the question of whether chromatin structure can also influence the outcome of CRISPR/Cas9 mutagenesis, for example, whether an insertion or deletion occurs at the target sequence. The results of the Weiss et al. study show that certain histone modifications do indeed have an influence here: Insertions occurred less frequently. The underlying mechanism was not investigated, but a direct or indirect influence on the DNA repair mechanisms, which repair the double strand break differently, is assumed.
Relevance of the results
The results of the Weiss et al. study show that the effectiveness of a CRISPR/Cas9 application in Arabidopsis is influenced by the chromatin structure of the genome. Other studies have come to opposite conclusions, namely that DNA in eu- and heterochromatin structures are equally well cut and modified with the gene scissors (Feng et al. 2016, Kallimasioti-Pazi et al. 2018, Yu et al. 2013). Contrasting studies suggest that CRISPR/Cas9 efficiency does not fail per se due to heterochromatin structures, but the influence of epigenetics is very complex (Přibylová et al. 2022).
Therefore, it can also be assumed that predictions regarding CRISPR/Cas9 insertions in bioinformatics programs developed so far are rather inaccurate. These programs mainly consider the DNA sequence; epigenetic data is as yet hardly ever included to determine the outcome of a CRISPR/Cas9 mutation.
Another interesting aspect of the publication is the attempt to increase the efficiency of CRISPR/Cas9 by bypassing the natural protective mechanisms of DNA. Here, this was accomplished by reducing DNA methylations. A previous study rebuilt chromatin structure by fusing CRISPR/Cas9 with a transcription activation domain, thus increasing the effectiveness of the gene scissors (Liu et al 2019). In future, it is likely that it will be possible to further maximize the efficiency of gene scissors, making DNA even more accessible, and thus bypassing more natural protective mechanisms.
Weiss T, Crisp PA, Rai KM, Song M, Springer NM, Zhang F. Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants. Plant Physiol. 2022 Jun 11:kiac285. doi: 10.1093/plphys/kiac285. Epub ahead of print. PMID: 35689624.
Feng C, Yuan J, Wang R, Liu Y, Birchler JA, Han F. Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System. J Genet Genomics. 2016 Jan 20;43(1):37-43. doi: 10.1016/j.jgg.2015.10.002. Epub 2015 Oct 30. PMID: 26842992.
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Liu G, Yin K, Zhang Q, Gao C, Qiu JL. Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhances Cas9 editing efficiency in vivo. Genome Biol. 2019 Jul 26;20(1):145. doi: 10.1186/s13059-019-1762-8. PMID: 31349852; PMCID: PMC6660936
Přibylová A, Fischer L, Pyott DE, Bassett A, Molnar A. DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner. New Phytol. 2022 May 6. doi: 10.1111/nph.18212. Epub ahead of print. PMID: 35524464.
Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics. 2013 Sep;195(1):289-91. doi: 10.1534/genetics.113.153825. Epub 2013 Jul 5. PMID: 23833182; PMCID: PMC3761309.