Structural Basis for CRISPR-Cas9 Mediated DNA Interrogation and Editing with 5' Truncated sgRNAs.

The CRISPR-Cas9 system has become the gold-standard for reliable genome engineering, yet the efficiency and accuracy of Cas9 targeting varies considerably across genomic targets. The system relies on RNA-based targeting of the Cas9 enzyme to a specific site in the DNA that harbors significant complementarity to the RNA guide. The off-target editing remains a persistent concern for implementing the Cas9 system in cells and clinical studies. The use of 5’ truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation in the complementarity region past 15 nucleotides prevents full Cas9 activation without compromising on-target binding. To better understand these mechanisms, we biochemically and structurally characterized Cas9 complexes formed with 5’ truncated sgRNAs. Using cryo-EM, we determined the structures of Cas9 bound to two different truncated sgRNAs. We find that reduced accessibility of the shorter non-target strand inhibits efficient nuclease lobe reorganization and can be overcome by modulating DNA topology. We also report on Cas9 complexes that enable more efficient cleavage with 5’ truncated sgRNAs and exhibit multi-turnover kinetics. Our findings provide a molecular basis for Cas9 targeting with 5’ truncated sgRNAs and support the development of new high-fidelity, multi-turnover Cas9 complexes in future studies.