Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

Abstract

The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Dictyostelium discoideum as a Model to Assess Genome Stability Through DNA Repair

“…Advances in mass spectrometry have recently revealed the major sites on histones that are ADPr in response to DNA damage (Jungmichel et al, 2013;Hottiger, 2015;Leidecker et al, 2016;Palazzo et al, 2018;Chen et al, 2021). The ability to manipulate multiple genes simultaneously and, in particular, to introduce mutations into endogenous histone proteins both singly and in combination (Rakhimova et al, 2017;Asano et al, 2021), offers a unique opportunity to study the role of these modifications in orchestrating DNA repair pathways.…”

“…This is especially relevant for studying the role of histones and how their post-translational modification at the site of DNA damage regulate DNA repair. D. discoideum contains single copies of genes encoding the majority of histone variants (Stevense et al, 2011;Hsu et al, 2012), allowing their disruption or mutation by gene replacement or genome editing by CRISPR/Cas9 (Muramoto et al, 2010;Hsu et al, 2012;Sekine et al, 2018;Iriki et al, 2019;Asano et al, 2021). In recent years a range of methods have been developed with which to study mutation rates (Saxer et al, 2012;Pontel et al, 2016) and specific repair pathways, including NHEJ, homologous recombination (HR; Hudson et al, 2005;Couto et al, 2011Couto et al, , 2013a and the posttranslational modifications that regulate these pathways, such as ADP-ribosylation (ADPr; Couto et al, 2011;Kolb et al, 2018).…”

CRISPR Toolbox for Genome Editing in Dictyostelium

“…A wide range of genetic techniques are available including homologous recombination-based methods (knockout, knock-in, and point mutation generation) and protein overexpression and expression of fusion-tagged proteins using various expression vectors (Gaudet et al, 2007;Veltman et al, 2009a). Genome editing using CRISPR/Cas9 has also been added to the toolbox for functional analysis in Dictyostelium, making it possible to modify genomes with higher efficiency than methods based on homologous recombination (Sekine et al, 2018;Iriki et al, 2019;Asano et al, 2021).…”

“…Using these various types of Cas9, a wide range of genome editing applications, including knockouts, inducible knockouts, knockdowns, knock-ins, point mutations and deletions, have been established in Dictyostelium (Table 1; Muramoto et al, 2019;Asano et al, 2021). SpCas9 is commonly used to create knockouts and has the highest efficiency among the available vectors, but the system is limited to applications in which genome editing does not need to be controlled temporally.…”

“…SpCas9 is capable of editing the target sequence in the presence of canonical "NGG" PAMs; however, due to the presence of AT-enriched regions in Dictyostelium genome, design of appropriate targets is elusive. To overcome this limitation, the newly engineered SpCas9 variants SpCas9-NG and SpRY were developed and are able to recognize a wider range of PAM sequences (Asano et al, 2021). This system provides valuable tools that will significantly expand the number of targetable gene loci available to generate mutants, even in AT-rich regions, with sufficient efficiency.…”