DNA-free genome editing

By Nitara Wijayatilake

CRISPR-Cas is a revolutionary gene-editing tool that has allowed applied molecular biologists to cut and manipulate genes more rapidly and efficiently than ever before.  Emmanuelle Charpentier and Jennifer Doudna won the Nobel Prize in Chemistry for their work on these incredibly versatile “molecular scissors” (Nobel Media, 2020). Scientists, encouraged by the momentum created by the Nobel winners, are using this technology in various ways to produce ground-breaking research. This essay will review a primary research paper on genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. The paper shows that DNA-free genome editing in this crop species is possible by delivering the Cas9-gRNA in a RNP (ribonucleoprotein) complex instead. This can produce high levels of mutagenesis in the plants as well as combatting some of the problems caused by DNA editing (Svitashev et al, 2016).

Double stranded breaks are really useful for gene editing. This is because of the way in which they are repaired. The first is NHEJ (Non-homologous end joining) which simply repairs the break using direct ligation, although this can be quite imprecise. The second is Homology directed repair which uses a homologous template to repair the break; this can also be in the form of an exogenously supplied foreign DNA that can be used for gene edits (Chang et al, 2017). CRISPR is really effective at producing these double stranded breaks, because, after being guided to the target sequence by guide-RNA, it uses its two nuclease domains to cut the DNA. Unfortunately, plant genome editing can be challenging, since plants have thick cell walls and penetration of it, to deliver nucleic acids or protein, can be difficult. Therefore, methods like electroporation are not possible. A biolistic delivery method is used to overcome this problem. In addition to this, using DNA is problematic because the DNA can integrate into the break and cause problems in the plant genome such as gene disruption or off-site cleavage. To mediate this problem, they use Cas9 and gRNA delivered in RNP complexes. 

Overall, the methods employed were particle bombardment of the pre-assembled Cas9, which had two nuclear localisation signals inserted into it, and gRNA in the form of a RNP complex, which had been mixed in a 1:2 molar ratio of the respective components. Specifically, four genomic regions in the maize were targeted: LIG, ALS2, MS26 and MS45. In the ALS2 gene, an edit was made using CRISPR where a homologous template was supplied that had a single nucleotide change coding for Serine instead of Proline. This change is known for conferring whole plant resistance to the herbicide, chlorsulfuron. Moreover, since the experiment investigates whether RNPs reduce off-target mutations, the off-site targets were identified using a Bowtie Sequence Aligner. Interestingly, it was found that RNP delivery reduced off-site cleavage significantly showing a 0.01% mutation frequency with RNP and 0.18% with DNA. Furthermore, the ALS2 gene edit was successful, tested by growing the maize cells on plates of chlorsulfuron as a test for direct selection. In general, the mutation frequency with the RNP delivery was very high, showing that it is a successful method of Cas9 and gRNA delivery.

In conclusion, the study was very successful in its delivery of RNA and CRISPR in the form of RNP complexes and the efficient mutagenesis of the maize cells. This research is extremely promising for the future of plant genome editing. Since no actual foreign DNA is inserted into the plant and, therefore, cannot integrate into the genome, there is no DNA modification. As there are strong fears surrounding GM crops, this would be an interesting way to alleviate some of the public’s worries whilst still advancing agricultural breeding practices in the future. Unfortunately, the research done in this paper does face some limitations as the particle bombardment method of delivery is not the most efficient. Since release of the paper, there has been successful production of maize protoplasts (Gao et al, 2019). A protoplast is a cell that has its cell wall removed by enzymatic digestion. With maize being such an essential crop and its production feeding so many people around the world, knowledge on this sort of gene editing possibilities is extremely important.

References:

Press release: The Nobel Prize in Chemistry 2020. (2020). NobelPrize.org. Available at: https://www.nobelprize.org/prizes/chemistry/2020/press-release/ [Accessed 28/10/2020]

Svitashev, S., Schwartz, C., Lenderts, B. et al. (2016).  Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7, 13274 https://doi.org/10.1038/ncomms13274

Chang, H., Pannunzio, N., Adachi, N. et al. (2017). Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol 18, 495–506 

https://doi.org/10.1038/nrm.2017.48
Gao, L., Shen, G., Zhang, L. et al.  (2019). An efficient system composed of maize protoplast transfection and HPLC–MS for studying the biosynthesis and regulation of maize benzoxazinoids. Plant Methods 15, 144 https://doi.org/10.1186/s13007-019-0529-2

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