Shivam Bhatt

Bio: Shivam Bhatt is an academic researcher from University of Nottingham. The author has contributed to research in topics: Transposase & Transposable element. The author has an hindex of 2, co-authored 3 publications receiving 21 citations.

Targeted DNA transposition in vitro using a dCas9-transposase fusion protein

Abstract: Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target-DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.

In vitro transposon targeting using a catalytically inactive Cas9

Abstract: Transposases are attractive tools for the integration of therapeutic transgenes into the chromosome for gene therapy applications. Typically, transgenes can be flanked with inverted-terminal repeat sequences, which are recognised by the transposase and integrated at random sites. Minimising detrimental insertions of transgenes is a key goal in the development of gene delivery vectors for gene therapy. We fused the Hsmar1 transposase to a catalytically inactive Cas9. Our aim was to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex. Although we could not detect any targeted transposition events in vivo, we achieved a 15-fold enrichment of transposon insertions into a 600-bp target site in an in vitro plasmid-to-plasmid assay. Additionally, we show that among those integrations that were successfully targeted, the location is tightly constrained to a site immediately to one side of the guide RNA target site. We present an in vitro proof-of-concept study demonstrating that the transposase insertion profile can be biased using a catalytically inactive Cas9 variant as a programmable DNA-binding module. One factor that limits the utility of this approach is that the transposon continues to integrate randomly. Although the dCas9 domain can be targeted to chromosomal lacZ, as evidenced by transcriptional repression, we were unable to detect any targeted insertions in the vicinity of the target site. Any targeted insertions that did occur were masked be a much larger number of random insertions. It is therefore necessary to develop a method for the temporal control of the transposase to allow Cas9 time to locate its target site.

Targeted DNA transposition using a dCas9-transposase fusion protein

Abstract: SUMMARY Homology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering.

Abstract: Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.

CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering

Abstract: Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR–Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA. However, the requirement for multiple expression vectors and low overall integration efficiencies, particularly for large genetic payloads, hindered the practical utility of the transposon. Here, we present a significantly improved INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting) system for targeted, multiplexed, and marker-free DNA integration of up to 10 kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplex insertions in three genomic loci, and facile multi-loci deletions when combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in other biomedically- and industrially-relevant bacteria, and developed an accessible computational algorithm for guide RNA design. This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering.