ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana .

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

CRISPR-Cas Systems* ; Gene Editing* ; Tetraploidy*; Plants, Genetically Modified/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Nicotiana/*genetics

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana . We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana . We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana . We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.

Plant Biotechnol J. 2020 Jan;18(1):45-56. (PMID: 31116473)
Mol Plant. 2018 Apr 2;11(4):631-634. (PMID: 29476918)
Mol Plant. 2021 May 3;14(5):722-731. (PMID: 33631420)
Mol Plant. 2021 Feb 1;14(2):352-360. (PMID: 33383203)
CRISPR J. 2018 Jun;1:239-250. (PMID: 31021262)
Plant Biotechnol J. 2020 Mar;18(3):770-778. (PMID: 31469505)
Mol Plant. 2018 Apr 2;11(4):627-630. (PMID: 29476916)
Biochem Cell Biol. 2017 Apr;95(2):187-201. (PMID: 28177771)
Nat Biotechnol. 2013 Aug;31(8):686-8. (PMID: 23929338)
Annu Rev Plant Biol. 2019 Apr 29;70:667-697. (PMID: 30835493)
Nat Plants. 2021 Jan;7(1):25-33. (PMID: 33398158)
Mol Biol Evol. 2015 May;32(5):1284-95. (PMID: 25637935)
Genome Biol. 2021 Jan 4;22(1):6. (PMID: 33397431)
PLoS One. 2021 Jan 14;16(1):e0245454. (PMID: 33444382)
Annu Rev Plant Biol. 2003;54:375-401. (PMID: 14502996)
Nat Plants. 2018 Mar;4(3):148-151. (PMID: 29483683)
Genome Biol. 2018 May 29;19(1):59. (PMID: 29807545)
Nat Biotechnol. 2020 Jul;38(7):883-891. (PMID: 32433547)
Plant Biotechnol J. 2019 Aug;17(8):1476-1478. (PMID: 30959555)
Plant Biotechnol J. 2018 Aug;16(8):1424-1433. (PMID: 29331077)
Nat Biotechnol. 2019 Mar;37(3):224-226. (PMID: 30809026)
Science. 2020 Jul 31;369(6503):566-571. (PMID: 32732424)
Plant Biotechnol J. 2019 Feb;17(2):499-504. (PMID: 30051586)

XDA24030404 Chinese Academy of Sciences Strategic Leading Science and Technology Project; 2019YFD1000201 National Key R&D Program of China

Keywords: ABE; ABE8e; CRISPR; PTG; STU; base editor

0 (RNA, Guide, CRISPR-Cas Systems)

Date Created: 20210602 Date Completed: 20210617 Latest Revision: 20240104

20240104

PMC8198424

10.3390/ijms22115663

34073486