Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9. Researchers identified that mutations in the WED domain of the engineered Cas9 enzyme, iGeoCas9, significantly enhance genome editing efficiency by accelerating DNA unwinding and expanding PAM preference. Cryo-EM structures revealed that iGeoCas9 forms stronger interactions with DNA substrates compared to wild-type GeoCas9. Biochemical analysis showed that iGeoCas9 accelerates DNA unwinding under magnesium-restricted conditions typical of mammalian cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, suggesting a general strategy for improving editing enzymes. The study highlights the role of the Cas9 WED domain in DNA unwinding and demonstrates how accelerated target unwinding improves Cas9-induced genome editing. The WED-domain mutations in iGeoCas9 also enable the enzyme to function at reduced magnesium concentrations, enhancing its activity in mammalian cells. The study further shows that these mutations can be transferred to other Cas9 proteins, such as Nme2Cas9, leading to improved genome-editing activities. The results indicate that optimizing the WED domain can lead to robust genome editors with expanded PAM compatibility and improved editing efficiency. The study also emphasizes the importance of understanding the structure-function relationship of Cas9 to improve genome editing technologies.Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9. Researchers identified that mutations in the WED domain of the engineered Cas9 enzyme, iGeoCas9, significantly enhance genome editing efficiency by accelerating DNA unwinding and expanding PAM preference. Cryo-EM structures revealed that iGeoCas9 forms stronger interactions with DNA substrates compared to wild-type GeoCas9. Biochemical analysis showed that iGeoCas9 accelerates DNA unwinding under magnesium-restricted conditions typical of mammalian cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, suggesting a general strategy for improving editing enzymes. The study highlights the role of the Cas9 WED domain in DNA unwinding and demonstrates how accelerated target unwinding improves Cas9-induced genome editing. The WED-domain mutations in iGeoCas9 also enable the enzyme to function at reduced magnesium concentrations, enhancing its activity in mammalian cells. The study further shows that these mutations can be transferred to other Cas9 proteins, such as Nme2Cas9, leading to improved genome-editing activities. The results indicate that optimizing the WED domain can lead to robust genome editors with expanded PAM compatibility and improved editing efficiency. The study also emphasizes the importance of understanding the structure-function relationship of Cas9 to improve genome editing technologies.