CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.