Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)—a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.