Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a.

Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation-a late transition state-and defies common descriptions of a "seed" region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.

Interactive youth science workshops benefit student participants and graduate student mentors.

Science communication and outreach are essential for training the next generation of scientists and raising public awareness for science. Providing effective science, technology, engineering, and mathematics (STEM) educational outreach to students in classrooms is challenging because of the need to form partnerships with teachers, the time commitment required for the presenting scientist, and the limited class time allotted for presentations. In our Present Your Ph.D. Thesis to a 12-Year Old outreach project, our novel solution to this problem is hosting a youth science workshop (YSW) on our university campus. The YSW is an interpersonal science communication and outreach experience in which graduate students from diverse scientific disciplines introduce middle and high school students to their cutting-edge research and mentor them to develop a white-board presentation to communicate the research to the workshop audience. Our assessment of the YSW indicated that participating young students expressed significantly more positive attitudes toward science and increased motivation to work in a STEM career after attending the workshop. Qualitative follow-up interviews with participating graduate students' show that even with minimal time commitment, an impactful science communication training experience can be achieved. The YSW is a low-cost, high-reward educational outreach event amenable to all disciplines of science. It enhances interest and support of basic science research while providing opportunities for graduate students to engage with the public, improve their science communication skills, and enhance public understanding of science. This YSW model can be easily implemented at other higher education institutions to globally enhance science outreach initiatives.

Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin.

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.