Better Base Editing in Plants
Better Base Editing in Plants

Better Base Editing in Plants

Adenine editing enzymes have been adapted for use in plants.

Feb 1, 2019
Ruth Williams
Base pair switch: CBEs convert C-G base pairs to T-A (top) by first deaminating the cytosine, converting it into a uracil, and then incorporating an adenine on the opposite DNA strand during replication or repair. ABEs convert A-T base pairs to G-C (bottom) by deaminating the adenine, converting it into an inosine, and then incorporating a cytosine on the opposite strand during replication or repair. Guide RNAs direct the CBE—which includes nuclease-free Cas9, cytosine deaminase, and uracil glycosylase inhibitor (to prevent uracil removal)—or ABE, which consists of nuclease-free Cas9 and adenosine deaminase, to the desired target sites in the plant genome.
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Altering the genetic code of crops and other plants to improve survival and yield, or to study physiology, has long been the pursuit of plant scientists and breeders. One of the newest methods for such genetic tinkering is called base editing, which utilizes modified components of the canonical CRISPR-Cas9 gene-editing system to make point mutations at specific nucleotides within the genome. The technique was originally developed by David Liu of Harvard University and colleagues for use in mammalian cells, but other scientists have recently adapted it for plants.

When using the traditional CRISPR-Cas9 system, researchers could choose between introducing mutations efficiently (by cutting the target gene, which creates random insertions or deletions during repair) or precisely (by introducing a DNA repair template containing a desired sequence change). The latter requires homology-directed repair, which tends to occur infrequently. Because base editing requires neither cutting nor templates, it can be both efficient and precise.

Scientists have been using cytosine base editors (CBEs), which convert C-G base pairs to T-A base pairs, in plants since 2017. Now, four groups of researchers, including Jin-Soo Kim and colleagues from the Institute of Basic Science in Daejeong, South Korea, have adapted adenine base editors (ABEs), which convert A-T to G-C, for use in plants too.

To make ABEs plant-friendly, “we optimized the codons [in the ABE expression vector] . . . and tested several plant promoters,” writes Kim in an email to The Scientist. The other groups employed similar optimization strategies.

Using their plant-tailored ABEs, Kim’s team altered one amino acid in the model plant Arabidopsis thaliana to convert it to a late-flowering variety, and altered a single splice site to create albino plants (Nat Plants, 4:427–31, 2018). “It was a proof-of-principle study,” Kim writes, and having shown that it works, “we are very excited.” Other researchers have used ABEs to produce herbicide-tolerant rice (Genome Biol, 19:59, 2018).

Plant scientist Alan Bennett of the University of California, Davis, who was not involved with any of these projects, is enthusiastic about the potential for base editing in plants. “It expands the toolkit for genome editing in plants, which is fantastic,” he says. “You now have more options, more opportunities to make very specific changes.”

(Nat Plants, 4:427–31, 2018; Mol Plant, 11:P627–30, 2018; Mol Plant, 11:631–34, 2018; Genome Biol, 19:59, 2018)

 Strategy
How it works
Possible genetic alterations
Efficiency
(% of alleles Converted)

Off-target mutations
Traditional
CRISPR-Cas9
A guide RNA targets a specific gene sequence and then recruits Cas9 nuclease, which cuts the DNA.
The DNA break can result in random insertions and deletions (indels) during repair, knocking out the gene. Or a template DNA can be used to introduce a desired sequence change with homology-driven repair.
High for indels—efficiency is
often greater than 50 percent

Low for homology-driven
repair—less than 1 percent
Apparently minimal, but more research needed
Base editing
Cas9 proteins lacking nuclease activity are fused to enzyme domains that can result in the conversion of cytosines to thymines or adenines to guanines. Guide RNAs then target these fusion proteins to the site of interest.
C-G to T-A or A-T to G-C
Between 5 percent
and 50 percent

Minimal. Less likely to be risky because the edit does not cut DNA