Unwinding matters: WED domain determines Cas9 activity by accelerating DNA unwinding and R-loop formation

Abstract

In a recent Cell paper,¹ Amy R. Eggers and colleagues from Jennifer A. Doudna's laboratory elucidated the mechanism by which an engineered CRISPR-Cas9 system significantly enhances editing efficiency by accelerating DNA unwinding and R-loop formation. The discovery and development of CRISPR-Cas9 gene editing technology have significantly revolutionized our ability to manipulate genomic sequences across a wide variety of species, as well as in clinical therapeutics. However, the efficiency of Cas9 proteins, especially the miniature ones, is generally low, requiring further improvement. In this article, the authors demonstrated that WED domain is a dominant determinant of target DNA unwinding and R-loop formation and its engineering improved genome-editing efficiency and expand PAM preference.

When binding to their target DNA sequences under the direction of PAM interaction and the guidance of single guide RNAs (sgRNA), Cas9 proteins separates the nontarget strand of the sgRNA from its target strand to form an R-loop structure. Subsequently, the cleavage activity domain of Cas9 cuts the DNA at this location, resulting in a double-strand break (Figure Fig 1). This process largely relies on the helicase activity of Cas9 to unwind the double-stranded target DNA into single strands for cutting. Evidence have shown that the formation of the R-loop is the rate-limiting step in this process.² Therefore, accelerating DNA unwinding and R-loop formation may be a potential strategy to improve CRISPR-Cas9 efficiency. Moreover, considering the size constraints imposed by certain delivery methods for in vivo applications, such as AAV, more compact Cas9 variants, including saCas9, cjCas9, and Nme2Cas9 have earned increasing interest. However, the activity of these editors generally lower than their larger orthologues, for example spCas9, which limited their applications.

In a previous study, the authors from the same lab evolved a Geobacillus stearothermophilus Cas9 (GeoCas9) using a bacterial double-plasmid selection system.³ The resulting evolved iGeoCas9 exhibited more than one hundred-fold improvement in the editing of mammalian genomes. However, its molecular basis remains unclear. Through cryo-electron microscopy structural analysis, the current study found that the three amino acids mutations in the WED domain significantly enhance the electrostatic interactions between iGeoCas9 and the DNA strand. This alteration may facilitate the separation of DNA strands and the formation of the R-loop before cutting. Additionally, the PAM recognition preference of iGeoCas9 is more relaxed compared to that of WT-GeoCas9. One possible hypothesis is that the mutations in the WED domain alter its interactions with the DNA backbone, leading to nonnative PAM recognition and cutting activity. To test this hypothesis, the authors conducted enzymatic activity assays using several mutants: GeoCas9 with only the WED domain mutation (R1), GeoCas9 without the WED domain mutation (KGR), the double mutant iGeoCas9, and the wild-type WT-GeoCas9. The results of these assays demonstrated that the mutations in the WED domain enhance Cas9 enzymatic activity and are critical factors in the relaxation of PAM recognition preference (Figure 1B). Consistent with these findings, a study from David R. Liu's lab in 2018 evolved the xCas9 variant with broad PAM compatibility using phage-assisted continuous evolution, resulting in seven mutations, six of which were in non-PI domains.⁴ Collectively, these studies provide compelling evidence that non-PI domains can influence PAM recognition. In an cleavage assay with suboptimal PAM and mismatched spacer, wild-type GeoCas9 and GeoCas9(R1), but not the iGeoCas9 and GeoCas9(KGR), showed an dependence of the cleavage on spacer mismatch. Noteworthy, mismatches adjacent to the PAM have been shown to improve the DNA unwinding and DNA cleavage kinetics of type II-C Cas9s.⁵ Therefore, these findings suggest that the mutations in the WED domain may enhance GeoCas9's DNA unwinding capability, thereby supersede the role of PAM in DNA target recognition.

Considering the kinetic aspects of Cas9 activity and its reliance on magnesium ions, the authors assessed the cleavage capabilities of WT-GeoCas9 and iGeoCas9 under magnesium-restricted conditions typical of mammalian cells. The results confirmed that, under low magnesium ion conditions, the WED domain mutations in iGeoCas9 accelerate R-loop formation compared with WT-GeoCas9, particularly in the mid-to-late stages (Figure 1C). This enhancement improves DNA unwinding ability, enabling the enzyme to maintain activity under low magnesium conditions, which is a prerequisite for gene editing in mammalian cells. This finding immediately suggested a practicable strategy to improve Cas9s activity via engineering the WED domain to enhance the DNA unwinding activity. Application of this strategy to another compact Cas9, Nme2Cas9, revealed that the resulting iNme2Cas9 has an accelerated DNA unwinding and R-loop formation activity, demonstrating gene editing activity significantly higher than those of WT Nme2Cas9 and comparable to spCas9.

In summary, unlike previous studies that focused on modifying other structural domains of Cas9 or the sgRNA, this study reveals the critical and unexpected role of the WED domain in regulating the gene-editing activity of type II-C Cas9. The increased interaction between the WED domain and target dsDNA markedly accelerates R-loop formation, allowing Cas9 to function effectively in mammalian environments with low magnesium ion concentrations. Besides, similar modifications in the WED domain across different Cas9 variants could also enhance editing efficiency. This discovery offers valuable insights into the structure-function relationship of Cas9 and provides a theoretical foundation for the optimization and design of more efficient and specific gene-editing tools. Future research could focus on further optimizing other functions of Cas9, such as enhancing its stability in various environments and expanding its target DNA sequence range. Additionally, integrating other gene-editing technologies, such as CRISPR-Cas12a or CRISPR-Cas13, could further increase the diversity and flexibility of gene editing. However, the activity of Cas9-derived editors such as base editors and prime editors is closely associated with their intrinsic activity, it remains to be further experimentally verified whether the mutations in the WED domain in this experiment are applicable to their derivative editors.

Mei Luo wrote the manuscript and drew the figure. ShaohuaYao supervised, and revised the manuscript writing. All authors have read and approved the final manuscript.

The authors declare no conflicts of interest.

The authors have nothing to report.