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The reduced genome-editing efficiencies of PAM-relaxed Cas9 variants (e.g., SpRY) are explained by two factors: (top) PAM-relaxed Cas9 variants display prolonged target search times, largely due to kinetic trapping at non-specific binding sites, which reduces the pool of free RNPs available for on-target identification. (Bottom) Upon reaching the target site, these variants also become kinetically trapped in a low-energy initial binding complex and unwind DNA slowly.

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