Scarless genome engineering

Overview

PBac transposons target genomic TTAA sites for insertion. The TTAA site is duplicated upon transposon insertion and restored to a single TTAA site upon excision. Selectable marker cassettes flanked by appropriate transposon ends can be similarly removed by PBac transposase. When placed in an endogenous TTAA site, cassette removal restores the genomic TTAA site for scarless removal of screenable markers following successful HDR. We have generated a collection of cassettes and vectors for rapid cloning of donor vectors for scarless genome engineering that are now available through the Drosohila Genomics Resource Center

 

pHD-ScarlessDsRed

pHD-ScarlessDsRed is a donor vector with a 3xP3-DsRed marker cassette flanked by PBac transposon ends. This vector can be used to generate targeted insertions, deletions, SNPs or any other modification with minimal locus disruption. Following DsRed-mediated identification of engineered lines, the marker cassette is easily removed through a single cross to PBac transposase. We've observed a high rate of cassette excision in the germline, with an average of 14% (2-30%) of progeny exhibiting precise excision of the marker. The image to the right shows the level of mosacism we typically see after crossing to transposase.

In the example below, a GFP tag is inserted into the start site of a target locus. The donor vector is designed such that after HDR, the Scarless-DsRed cassette is inserted into a nearby TTAA site in the adjacent intron. After recovery, the Scarless-DsRed cassette is removed leaving behind only the GFP tag. 

 

The pHD-ScarlessDsRed (map below) plasmid can be used for Gibson- or enzyme-based generation of donor plasmids for scarless gene editing. It's available at the Drosophila Genomics Resource Center (DGRC # 1364).

 

 

pHD-sfGFP-ScarlessDsRed, pHD-3xFLAG-ScarlessDsRed and pHD-2xHA-ScarlessDsRed

These vectors are designed for simplified cloning of donor vectors for endogenous tagging with sfGFP, 3xFLAG or 2xHA. Each tag is flanked on the 5' and 3' side by a linker sequence so that they can be used of N-terminal, C-terminal or internal tagging. The PBac-flanked 3xP3-DsRed cassette is in a TTAA sequence that has been included in the 3' linker sequence making the tag and the scarless marker a single cassette that can be easily cloned into donor vectors. Once the ScarlessDsRed marker is removed using PBac transposase, only the linker and tag sequences are left in the target locus. 

In the example below, the pHD-sfGFP-ScarlessDsRed cassette is used to insert sfGFP into the n-terminus of a target gene. After HDR events have been identified, the ScarlessDsRed marker is removed leaving only the sfGFP tag and linker sequences.

 

 

Mosaic Endogenous Tagging

One attractive application of our new tagging vectors (pHD-sfGFP-ScarlessDsRed, pHD-3xFLAG-ScarlessDsRed and pHD-2xHA-ScarlessDsRed) is endogenous tagging of target genes in specific or random cell types. Before the ScarlessDsRed marker is removed, the locus is disrupted. Once the marker has been removed, the tagged gene product can be produced. Ubiquitous expression of PBac transposase can cause removal of the marker in random cells for mosaic analysis. Alternatively, cell specific expression of the transposase can be used to restrict expression of the tagged gene product in specific cell types.

The image below, ubiquitous expression of transposase was used to create a mosaic animal in which a random subset of cells express the tagged gene product. Open arrow heads indicate cells that still have the PBac-based marker disrupting the target locus interrupting the expression of the tagged gene product. Arrows indicate cells where the ScarlessDsRed cassette has been removed allowing expression of the GFP-tagged gene product.

 

pHD-sfGFP-ScarlessDsRed (DGRC #1365), pHD-3xFLAG-ScarlessDsRed (DGRC #1367) and pHD-2xHA-ScarlessDsRed (DGRC #1366) can be used for rapid Gibson-based generation of donor plasmids for scarless tagging of endogenous genes.

                       

 
Click on plasmid names for sequences in ApE format. ApE is available from M. Wayne Davis through the Jorgensen lab here.