What tool or method do you wish existed?

We asked researchers from a range of disciplines across biology, engineering, and medicine to describe a current technological need. The goal is to provide a sample of the various technological gaps that exist and inspire future research projects.

Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon.

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Systematic discovery of DNA-binding tandem repeat proteins.

Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.

LncRNA-Smad7 mediates cross-talk between Nodal/TGF-ß and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells.

Transforming growth factor ß (TGF-ß) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-ß and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-ß and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage.

Cas12a is one of the most commonly used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its target among numerous nonspecific and off-target sites. Cas12a utilizes one-dimensional diffusion along the contour of dsDNA to efficiently search for its target. However, due to a lack of structural information of the transient diffusing complex, the residues mediating the one-dimensional diffusion of Cas12a are unknown. Here, combining single-molecule and ensemble assays, we found that nonspecific interactions between Cas12a and dsDNA at the PAM downstream cause asymmetric target search regions of Cas12a flanking the PAM site, which guided us to identify a positive-charge-enriched alpha helix in the REC2 domain serving as a conserved element to facilitate one-dimensional diffusion-driven target search of AsCas12a, LbCas12a and FnCas12a. In addition, this alpha helix assists the target cleavage process of AsCas12a via stabilizing the cleavage states. Thus, neutralizing positive charges within this helix not only significantly slows target search but also enhances the specificity of AsCas12a both in vitro and in living cells. Similar behaviors are detected when residues mediating diffusion of SpCas9 are mutated. Thus, engineering residues mediating diffusion on dsDNA is a new avenue to optimize and enrich the versatile CRISPR-Cas toolbox.

The compact Casπ (Cas12l) 'bracelet' provides a unique structural platform for DNA manipulation.

CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.

A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis.

Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system remodels has been poorly understood. Here we identify a new type of membrane contact formed between the ER-Golgi intermediate compartment (ERGIC) and the ER-exit site (ERES) in the ER-Golgi system, which is essential for promoting autophagosome biogenesis induced by different stress stimuli. The ERGIC-ERES contact is established by the interaction between TMED9 and SEC12 which generates a short distance opposition (as close as 2-5 nm) between the two compartments. The tight membrane contact allows the ERES-located SEC12 to transactivate COPII assembly on the ERGIC. In addition, a portion of SEC12 also relocates to the ERGIC. Through both mechanisms, the ERGIC-ERES contact promotes formation of the ERGIC-derived COPII vesicle, a membrane precursor of the autophagosome. The ERGIC-ERES contact is physically and functionally different from the TFG-mediated ERGIC-ERES adjunction involved in secretory protein transport, and therefore defines a unique endomembrane structure generated upon stress conditions for autophagic membrane formation.

Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search.

RNA-guided Streptococcus pyogenes Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151-1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both in vitro and in vivo applications of Cas9.