Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases

Abstract

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5-prime nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.

CvkR, a novel MerR-type transcriptional regulator, is a repressor of class 2 type V-K CRISPR-associated transposase systems

“…Native type V-K CAST systems have thus far only been found in certain cyanobacteria 11, 14 . Their better characterization is of fundamental interest, and these systems hold great promise for the development of novel genome editing tools 28 . The primary function of native CRISPR-Cas systems is defense against mobile genetic elements.…”

Genome engineering by RNA-guided transposition forAnabaenaPCC 7120

“…Unidirectional insertion of the cargo (Figure 5) is consistent with the unidirectional formation of the polymeric TnsC complex with the target DNA and Cas12k serving to recruit the TnsB transposase at the target site (Park et al, 2021; Querques et al, 2021). Co-integration of the plasmid sequences supplied along with the transposon cargo was reported to occur at frequencies ranging from some 85% (Vo et al, 2020; Vo et al, 2021) to 19.4%, and 0,6% in the specific case of the Cas12k-homing endonuclease fusion (Tou et al, 2022). An explanation for co-integration was also proposed: the CAST lacks the TnsA required to excise the transposon and the Cas12k lacks the endonuclease activity to substitute for the TnsA.…”

Transposons and CRISPR: Rewiring Gene Editing

“…In the second approach, Cas12k has been fused to either TniQ or TnsC and the insertion efficiency was measured. However, none of the constructs tested yielded an efficiency higher than that of the separate components . This could be due to the wrong orientation of the proteins imposed by the design of the construct, misfolding, steric impediment, etc.…”

“…Protein engineering has been used in two ways to optimize subtype V-K. First, additional components have been added to the excision and integration system to avoid cointegrates, and second, protein fusions have been designed to simplify the targeting system. 86 In the first approach, given that subtype V-K lacks TnsA, a high percentage of its transposition products are cointegrates. In an effort to supply TnsA's function, Tou et al 86 fused TnsB to a homing endonuclease mutant with specific ssDNA nickase activity (nAniI).…”

“…86 In the first approach, given that subtype V-K lacks TnsA, a high percentage of its transposition products are cointegrates. In an effort to supply TnsA's function, Tou et al 86 fused TnsB to a homing endonuclease mutant with specific ssDNA nickase activity (nAniI). These combinations led to a dramatic decrease in the level of cointegrate products from 20% to <1% when using target sequences present in plasmids and 3% when using genomic target sequences.…”