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.

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.

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.

Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays-with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following R-loop formation, both DNA strands are cleaved by the RuvC nuclease. Kinetic characterization of Cas12a DNA targeting showed R-loop formation is rate-limiting for cleavage and the specificity-determining step. Ternary structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and how R-loop length translates to nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop structures to elucidate how Cas12a forms its 20bp R-loop and delivers DNA into the RuvC active site. We show that Cas12a first interrogates 5bp of target DNA using a 'kinetic seed,' followed by dramatic Rec domain mobility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec domains to make substantial contacts throughout the majority of the R-loop. To describe how R-loop completion is linked to DNA cleavage, we propose a 'base grabbing' mechanism in which the RuvC lid engages via base stacking with the nontarget strand traversing the RuvC site to trigger displacement and active site exposure. Biochemical interrogation shows that lid resetting is required prior to target strand cleavage. Our kinetics-guided structure determination provides a comprehensive model describing Cas12a DNA targeting and underscores mechanistic differences between Cas12a and Cas9.