CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.
CRISPR/Cas-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (SNPs) that require precision genome editing tools for correction of the sequences. Although homology-directed repair (HDR) of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.
The PUGS scale published in 2017 and cited below included Figure 1, a version of the scale that assesses anticipated uncertainties from genome sequencing prior to return of results. Data from this scale were used for confirmatory factor analyses that led to the PUGS being reduced from a 10-item to an 8-item scale. A version of the PUGS that assesses uncertainties following return of sequencing results was inadvertently omitted from the manuscript. In its place appears a duplicate of Figure 1. The sentence on page 6 should have read: "At the 1-month follow-up the eight-item PUGS was further modified to reflect that results had been returned. This version of the PUGS intended for use upon return of sequencing results can be found in Figure 3."
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