Document Type

Article

Publication Date

10-31-2019

Keywords

JGM

JAX Source

Nat Commun 2019 Oct 31; 10(1):4968

Volume

10

Issue

1

First Page

4968

Last Page

4968

ISSN

2041-1723

PMID

31672965

DOI

https://doi.org/10.1038/s41467-019-12891-2

Grant

HG009900,CA034196

Abstract

Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.

Comments

KOLF2-C1 cells were a gift from Bill Skarnes and were derived from the HipSci consortium. We gratefully acknowledge the contribution of the Flow Cytometry, Cell Engineering Services at The Jackson Laboratory for expert assistance with this publication. Special thanks to our Research Program Development group for assistance with the editing of the manuscript.

This open access article is licensed under a Creative Commons Attribution 4.0 International License.

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