Sammanfattning

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

Nyckelord

CRISPR-Cas9; moss; multiple gene targeting; butenolide receptor; KAI2; AP2/ERF transcription factor

Publicerad i

G3
2016, Volym: 6, nummer: 11, sidor: 3647-3653

UKÄ forskningsämne

Biokemi och molekylärbiologi


Publikationens identifierare

DOI: https://doi.org/10.1534/g3.116.033266

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/84591