Structural basis for target site selection in RNA-guided DNA transposition systems

CRISPR-associated transposition systems allow guide RNA–directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo–electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate–bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

Structural basis for DNA targeting by the Tn7 transposon

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP-hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

Structural basis for target-site selection in RNA-guided DNA transposition systems

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, structures with the transition state mimic, ADPᐧAlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

Comparative Study between the CRISPR/Cpf1 (Cas12a) and CRISPR/Cas9 Systems for Multiplex Gene Editing in Maize

Although the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been proved to be an efficient multiplex gene editing system in maize, it was still unclear how CRISPR/Cpf1 (Cas12a) system would perform for multiplex gene editing in maize. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. The bZIP transcription factor Opaque2 (O2) was used as the target gene in both systems. We found that in the T0 and T1 generations, the CRISPR/Cpf1 system showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize and is a valuable alternative choice for gene editing in crops.

The Integration Preference of Sleeping Beauty at Non-TA Site Is Related to the Transposon End Sequences

Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.

Comparative Study Between the CRISPR/Cpf1 and CRISPR/Cas9 systems for Multiplex Gene Editing in Maize

Background: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully used for multiplex gene editing in crops. Although CRISPR/Cas9 system has been proved to be an efficient multiplex gene editing system in crops, it was still unclear how CRISPR/Cpf1, a natural direct repeat (DR)-based multiplex gene editing system, performed in crops. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. Results: The bZIP transcription factor Opaque2 (O2) was used as the target gene to evaluate the editing efficient of both systems. We found that in the T0 generation, the CRISPR/Cpf1 system showed low editing efficiency with only one mutation, while the CRISPR/Cas9 system generated many different types of on-target mutations. In the T1 generation, the CRISPR/Cpf1 system still showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. Conclusions: In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize. Thus, the CRISPR/Cpf1 system is a valuable alternative choice for gene editing in crops.

The integration preference of Sleeping Beauty at non-TA site is related to the transposon end sequences

Background: Sleeping Beauty (SB) transposon had been thought to strictly integrate into TA dinucleotides. Recently, we found that SB also integrates into non-TA sites at a lower frequency. Here we performed further study on the non-TA integration of SB. Results: 1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines. 2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites. 3) The consensus sequence of the non-TA target sites only occur at the opposite side of the sequenced junction between transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations. 4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. When the transposon end sequence is mutated, the consensus sequences changed too. Conclusion: The interaction between the SB transposon end and genomic DNA may be involved in the target site selection of the SB integrations at non-TA sites.

PRC1 catalytic activity is central to Polycomb system function

SummaryThe Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how its target genes are selected and whether its histone modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. We demonstrate that a mutation widely used to disrupt PRC1 catalysis is hypomorphic, complicating the interpretation of previous studies. To overcome this, we develop a new inducible mutation system in embryonic stem cells that completely ablates PRC1 catalytic activity, revealing that catalysis by PRC1 drives Polycomb chromatin domain formation and higher-order chromatin interactions. In the absence of catalysis, we uncover the primary DNA-based targeting determinants that direct Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling new evidence supporting a PRC1-initiated pathway for Polycomb system function in gene regulation.