Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

Abstract

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells

“…In the groups with puromycin selection, the T7E1 assay detected 12.9–72.1% indels, which was similar to the results of the previous reports ( Abu-Bonsrah et al, 2016 ; Bai et al, 2016 ; Antonova et al, 2018 ). The performance of T7E1 may be impacted by the length and identity of base pair mismatches, flanking sequence, secondary structure, and relative abundance of mutant sequence ( Vouillot et al, 2015 ; Michalski et al, 2021 ). Several reports showed that the indel frequencies of T7E1 assays were significantly less than NGS assays when the amount of edited DNA is a trace or abundant ( Santhakumar et al, 2018 ; Sentmanat et al, 2018 ; Jin et al, 2020 ).…”

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

“…Michalski et al. (2021) augmented the Cas9 gene with monocot codon‐optimized human THREE PRIME REPAIR EXONUCLEASE 2 (TREX2) sequence to improve nuclease activity in hexaploid triticale ( x Triticosecale ; 2 n = 6 x = 42) protoplasts and observed increases in mutagenesis efficiency of up to 19‐fold for gRNAs expressed from TREX2‐enhanced vectors.…”

“…Co-editing of The Plant Genome multiple alleles was identified in 54% of potato transformants containing the dMac3-Cas9 system, while only 7% of transformants containing the ADH enhancer carried mutations in multiple target alleles (Kusano et al, 2018). Michalski et al (2021) augmented the Cas9 gene with monocot codon-optimized human THREE PRIME REPAIR EXONU-CLEASE 2 (TREX2) sequence to improve nuclease activity in hexaploid triticale (x Triticosecale; 2n = 6x = 42) protoplasts and observed increases in mutagenesis efficiency of up to 19-fold for gRNAs expressed from TREX2-enhanced vectors.…”

Somatic Embryogenesis, Genetic Modification, and GMOs at the Department of Plant Biotechnology and Cytogenetics at the IHAR

“…In a short time, MAH 1590 was registered by Dr. Bogusław Łapiński as 'Bogo, ' a new high-yielding winter triticale variety. 'Bogo' has been successfully grown for many years in Poland, and aer 30 years since our discovery, it continues to serve as a model genotype used in studies on in vitro plant regeneration and transformation of triticale (Hensel et al, 2012;Michalski et al, 2021;Oleszczuk et al, 2004;Sowa et al, 2005).…”

“…Being aware of all these challenges, we continuously master genomic techniques through scientific research and must be ready to produce improved and safe breeding materials, now by optimizing the recent CRISPR/Cas9 method (Michalski et al, 2021).…”