Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools

Abstract

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations.

Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter

“…The Cas9 rice expression vector (pUbi-Cas9) [ 32 ] and the sgRNA expression vector (pENTR-sgRNA) under the control of the OsU6 promoter [ 33 ] were used to construct the pCas9/OsSWEET14-gRNA expression vector. The complementary oligonucleotides with appropriate 4-bp overhangs were synthesized by Macrogen (Korea).…”

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

“…Precise genome modification overcomes the difficulties of traditional random uncontrolled mutagenesis and unpredictable insertions into the plant genome and thus has potential positive impacts on plant breeding (Schiemann et al, 2019). As a potential drawback, the CRISPR-Cas technology can generate undesirable Frontiers in Plant Science | www.frontiersin.org 9 off-targets but, although frequently predicted in silico, has been shown to occur rarely in plants (Young et al, 2019;Graham et al, 2020;Herbert et al, 2020;Gao et al, 2020a).…”

Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens

“…(AsCas12a), and Francisella novicida (FnCas12a) have been the most commonly used enzymes in several plant species. They generally display higher specificity and less or no off-targets compared with Cas9 ( Endo et al., 2016 ; Begemann et al., 2017 ; Kim et al., 2017 ; Tang et al., 2017 , 2018 , 2019 ; Xu et al., 2017 , 2019a ; Yin et al., 2017 ; Li et al., 2019a ; Lee et al., 2019 ; Herbert et al., 2020 ).…”

“…The development of such tools in plants would be of great interest, with the possibility of anticipating NHEJ-mediated DSB repair outcomes for predictable mutations in coding or regulatory sequences. While the Cas9 nuclease mainly generates small indels ( Figure 2 A), Cas12a and Cas12b predominantly produce larger deletions ( Bernabe-Orts et al., 2019 ; Herbert et al., 2020 ). Whether these different mutation footprints are the result of Cas12 cleavage properties and/or due to the binding time of the nuclease to the broken DNA is still unclear ( Chen et al., 2018 ; Que et al., 2019 ).…”

“…Whether these different mutation footprints are the result of Cas12 cleavage properties and/or due to the binding time of the nuclease to the broken DNA is still unclear ( Chen et al., 2018 ; Que et al., 2019 ). Regardless of their mechanisms, the cleavage properties of Cas12 enzymes could have specific practical interest compared with Cas9 such as the removal of larger coding or regulatory motifs ( Li et al., 2020e ; Herbert et al., 2020 ).

Figure 2 NHEJ- and HR-Mediated DNA Mutations after CRISPR Cleavage.

…”