Novel molecular requirements for CRISPR RNA-guided transposition

Novel molecular requirements for CRISPR RNA-guided transposition

ABSTRACT

CRISPR-associated transposases direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type One-F Vibrio cholerae CRISPR-associated transposase system. On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor. Remarkably, we found that VchCAST requires integration host factor for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CRISPR-associated transposase systems.

GRAPHICAL ABSTRACT

INTRODUCTION

Transposons are pervasive genetic elements capable of mobilizing between distinct genetic contexts using various targeting pathways, which serve as a potent force for genome evolution in all domains of life. Despite their abundance and sheer diversity, most transposons share a common feature in the presence of inverted repeat sequences that dictate the boundaries of the mobile element. These terminal transposon sequences are referred to as the left and right ends and typically encode one or more binding sites recognized by oligomeric transposase proteins through specific protein-DNA interactions.

DNA transposons (Class Two) may be classified by their hallmark transposase domain, leading to major classes of DD(E/D) transposons, serine transposons, tyrosine transposons and Y one/Y two-transposons. DD(E/D)-family transposable elements encompass all known examples of 'cut-and-paste' DNA transposons, which are excised from the donor site and inserted into the target site. This reaction relies on mechanistic and enzymatic symmetry, with transposase subunits executing identical chemical steps on both transposon ends that involve coordinated nucleophilic attacks during strand cleavage and joining. The transposase binding sites themselves, however, are not always positioned symmetrically. Whereas some transposons encode symmetrically-positioned transposase binding sites within identical left and right ends, including Tc one/mariner-family transposons such as Mos one, other elements encode asymmetrically-positioned binding sites within distinct left and right ends, including Hermes, piggy-Bac, Bacteriophage Mu, P elements, and Tn seven-family transposons. For the well-characterized Tn seven transposon, the asymmetric transposon ends facilitate strict control over integration orientation, allowing one transposon end to be preferentially integrated adjacent to a target site, but the underlying mechanistic basis explaining this preference is unresolved.

Tn seven-family transposons exhibit a modular nature in that they encode conserved core transposition proteins-TnsA, TnsB and TnsC-but diverse target site selection components. TnsB is a DD(E/D)-family integrase that recognizes and binds the transposon ends, catalyzes three prime cleavage of both transferred strands at the donor site, and catalyzes the transesterification reaction at the target site. TnsA is an endonuclease protein that forms a heteromeric complex with TnsB and cleaves the five prime end of the non-transferred strand, collectively allowing for full excision of the transposon from the donor site. TnsC is an AAA+ ATPase protein that communicates between the transposase module and the targeting component. Tn seven-like transposons encode a sequence-specific DNA-binding protein as their targeting module, often of the TniQ family and referred to as TnsD, which directs transposition to a safe-harbor locus such as glmS, comM, yhiN and par E. In contrast, CRISPR-associated transposons use nuclease-deficient CRISPR-Cas systems to catalyze programmable, RNA-guided DNA transposition.

We previously reported RNA-guided transposition activity for VchCAST, a Tn seven-like transposon from Vibrio cholerae (also referred to as Tn six thousand six hundred seventy-seven), which mediates efficient and highly specific DNA integration in an Escherichia coli heterologous host. VchCAST encodes a Type One-F CRISPR-Cas system that specifies integration sites through RNA-guided DNA targeting by a multi-subunit complex called Cascade. Importantly, Cascade binds DNA in complex with an accessory transposition protein, TniQ, which ultimately recruits TnsC and the heteromeric TnsAB transposase, in complex with the donor DNA, to assemble the catalytically active transpososome. DNA integration occurs roughly fifty base pairs downstream of the R-loop formed by TniQ-Cascade, but the preferred transposon insertion site varies across separate targets, with the dominant integration position ranging between forty-six to fifty-two base pairs downstream of the target site. The sequence determinants underlying these heterogeneous integration distances remained enigmatic in our previous work. Additionally, the role and requirement for multiple putative TnsB binding sites within both transposon ends is unclear, limiting efforts to further engineer VchCAST as a DNA integration tool. Like other Tn seven-family transposons,

the transposon left and right ends encode asymmetrically positioned TnsB binding sites, which may also relate to the biased orientation with which transposon insertions occur.

Here, we employ library-based experiments in combination with high-throughput sequencing to investigate DNA sequence requirements during RNA-guided transposition by VchCAST. By individually mutating both transposon ends and measuring resulting DNA integration activity, we revealed sequence requirements of transposase binding sites that also mediate transposase-transposon cognate specificity. Interestingly, our results indicated that the relative positioning of each transposase binding site plays a crucial role in defining the proper architecture of the transposome complex, with spacing patterns that correspond to the helical pitch of double-stranded DNA. These mutational data also revealed the importance of an integration host factor binding site within the left transposon end, and subsequent genetic knockout and rescue experiments confirmed the role of integration host factor in stimulating transposition efficiency in E. coli. Finally, we uncovered sequence preferences at the site of integration, and we exploited our mutagenesis data to rationally engineer the transposon right end to enable in-frame tagging of endogenous protein-coding genes. Collectively, this work expands our understanding of both protein and DNA sequence requirements of Tn-seven-like transposons, reveals insights into the architecture of the transposome complex, and provides new knowledge to inform the design of custom transposon sequences for genome engineering applications.