High-throughput screens utilizing the CRISPR-SpCas9 system enable detailed analysis of cis-regulatory sequences within their endogenous chromatin context, which is crucial for appropriate gene regulation. However, the requirement for a specific protospacer adjacent motif (PAM), such as the NGG PAM (~1/16) for SpCas9, has largely limited the resolution, making many regulatory elements and pathogenic variants inaccessible to manipulation. 

In a study published in Cell Genomics, researchers led by Prof. XUE Chaoyou from the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, together with Prof. RAO Shuquan from Institute of Hematology & Blood Diseases Hospital of the Chinese Academy of Medical Sciences & Peking Union Medical College, adopted SpRY to identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. 

Researchers found that although saturated SpRY mutagenesis screens can accurately identify functional regulatory elements, they are unable to interrogate variants at single-base resolution due to the fact that some sgRNAs have low to nearly zero editing efficiency at certain sites.  

To overcome this, researchers adopted SpRY-ABE and SpRY-CBE base editing, which allows an editing window of up to five base pairs for each functional sgRNA, compensating for the non-functional sgRNAs to achieve single-base resolution. 

Although SpRY has significantly expanded the targeting region, researchers observed a very broad editing efficiency (0%-70%) and much lower average editing efficiency than expected at genomic sites with their preferred PAMs. In addition, given the evolved role of PAMs in facilitating target searching kinetics, it remains unclear how SpRY quickly locates its targets and whether the broad editing efficiency is related to the target searching kinetics. 

Therefore, researchers combined experiments, simulations, and theoretical analyses to investigate how SpRY searches for its targets. They found that SpRY exhibits a preference for the seed region for efficient targeting, and highlighted the importance of considering Cas9 target searching kinetics in the successful engineering of PAMless SpCas9 variants.  

This study, published in Cell Reports, provides valuable insights for further PAMless Cas9 protein engineering research efforts.