Effective transformations result in an efficient HDR DNA repair system in poplar

Published: 5 April 2022| Version 2 | DOI: 10.17632/5tp44bnczy.2
Contributors:
Ali Movahedi,
Hui Wei,
Ali Kiani-Pouya,
Fatemeh Rasouli,
Saeid Kadkhodaei,
,
Liming Yang,
Qiang Zhuge

Description

Genetic improvements and RNA-guided genome editing of plants use clustered regularly interspaced short palindromic repeats (CRISPR) associated with a potential nuclear-localized endonuclease (Cas9). DNA system repairs include homology-directed repair (HDR) and non-homologous end-joining (NHEJ). HDR is often used for replacing nucleotides with one donor DNA template (DDT), including homologous sequences, while NHEJ repairs damaged DNA by inserting or deleting (Indel) nucleotides in DSB regions. We recruited CRISPR-Cas9 and designed three variables, Agrobacteria inoculator concentration, the ratio of pDDT/pgRNA, and homologous arm length, to integrate nptII and 2XCamV35S into the MKK2 promoter zone. Here, we showed that recovered events on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by precise integrated 2XCamV35S and nptII, improving biochemical and phenotypic properties. Our data verified efficient transformation achieved by Agrobacterium inoculator OD600=2.5, increased DDTs numbers among cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused to occur efficient HDR leading to more MKK2 expression.

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Steps to reproduce

Efficient transformation methods have been used to knock in exogenous nptII and promoter 2XCamV35S leading to occur efficient HDR.

Institutions

Nanjing Forestry University

Categories

DNA Transformation, Efficiency Analysis, Genome Editing

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