Protocols: pHD-DsRed-attP dsDNA donor cloning

Rapid dsDNA donor cloning with the pHD-DsRed-attP vector

 

pHD-DsRed-attP (available soon from Addgene) is a vector for making dsDNA donor templates for homology-directed repair (HDR). This vector is designed for replacing a targeted locus with a 50-bp attP phage recombination site and is positively marked with a removable (floxed) 3XP3-dsRed construct for screening. It has 2 multiple cloning sites (MCS) for inserting homology arms that immediately flank the targeted locus. Work from Dana Carroll’s lab indicates that homology arms of ~1Kb mediate efficient HR (Beumer et al., 2013).

pHD-DsRed-attP Map

 

Step 1. Homology arms

Homology arms should contain sequence immediately adjacent to your cleavage sites for efficient HDR. In the example below two flanking CRISPR gRNAs are used to generate a defined deletion of a target locus. The intervening locus can be replaced with attP by incorporating homology arms immediately flanking each cleavage site into pHD-DsRed-attP. Note that the cleavage sites are within the CRISPR target sites and the portion of the target sequence that will remain after cleavage should be included in each homology arm.

example:

Homology Arm Extent

 

Step 2. Primer design for homology arm 1 (AarI)

In addition to traditional restriction enzyme sites, the pHD-DsRed-attP MCSs contain type IIs restriction sites (AarI for homology arm 1 and SapI for homology arm 2) for seamless and convenient cloning. Use of the type IIs sites requires precise primer design defined below.

AarI (site: CACCTGC) is a 4/8 cutter meaning it creates a 4-bp 5’ overhang 4-bp downstream of the cut site (illustrated as red lines). Primers should contain extra 5’ sequence (Ns) and 4-bp of “filler” sequence (Ns) followed by the appropriate overhang.

 

 

Step 3. Primer design for homology arm 2 (SapI)

SapI (site: GCTCTTC) is a 1/4 cutter meaning it creates a 3-bp 5’ overhang 1-bp downstream of the cut site (illustrated as red lines). Primers should contain extra 5’ sequence (Ns) and 1-bp of “filler” sequence (Ns) followed by the appropriate overhang.

 

Download a PDF of this page