Kovo 25 d. 10:00 val. R 201 auditorijoje biofizikas Marius Rutkauskas skaitys paskaitą "A random-walk in DNA recognition: Off-target discrimination by the CRISPR-Cas Cascade surveillance complex" (santrauka anglų k. apačioje).

 

 

 

Abstract: CRISPR–Cas systems constitute adaptive immune mechanisms that are widely distributed among prokaryotic organisms, including bacteria and archaea. These systems function through the sequence-specific recognition and degradation of foreign genetic material originating from invading bacteriophages and other mobile genetic elements. After it was discovered that the target specificity of CRISPR–Cas effector and surveillance complexes can be reprogrammed through the design of guide RNAs, these systems rapidly became indispensable tools in genome engineering and gene editing, as well as in a broad range of biomedical applications, including gene therapy and functional genomics. Despite their remarkable versatility and efficiency, it has become evident since their initial implementation in genome engineering that CRISPR–Cas systems can exhibit significant off-target activity. Such activity arises from the ability of CRISPR-associated complexes to recognize and cleave DNA sequences that are only partially complementary to the guide RNA, thereby posing challenges for precise genome manipulation.

In the present study, the process of R-loop formation was investigated using single-molecule magnetic tweezers. An R-loop is a nucleic acid structure that is formed during target recognition through the hybridization of the CRISPR RNA (crRNA), carried by the Cascade surveillance complex, with its complementary DNA target strand. By applying magnetic tweezers, the dynamics of R-loop formation were directly observed on both fully complementary DNA targets and targets containing mismatches. The datasets obtained from these single-molecule measurements provide insight into the fundamental molecular mechanism underlying target recognition by the Cascade complex. Furthermore, intrinsic limitations associated with attempts to enhance target specificity through Cascade engineering were identified. It was additionally demonstrated that the complex molecular process of R-loop formation can be quantitatively described by a relatively simple biophysical model.

Overall, this study contributes to a deeper understanding of the molecular principles that govern antiviral defense mediated by CRISPR–Cas systems and provides a conceptual framework that may facilitate the development of more accurate computational tools for predicting off-target activity in genome editing applications.