Introduction

The CIS display, similarly to Ribosome-, mRNA- and cDNA-displays, is based on in vitro protein synthesis with one significant advantage: newly translated protein get immediately connected to the DNA encoding (https://www.pnas.org/content/101/9/2806). Two idea was behind its development: to avoid relying on prone for degradation mRNA in the selection process and to keep the high level of genetic diversity similar to the mRNA and Ribosome displays (10¹³). Other DNA-based in vitro selection methods are largely based on encapsulation of DNA into vesicles which needs to be clonal and therefore genetic libraries get severy dilluted with maximum coverage 10⁹ mutants. The CIS display reportedly allows recovery of a specific binder from a pool of non-binding mutants present at a ratio of 1 in 10¹⁰!

Principle of CIS display

In the CIS display a linear dsDNA served as a template. It usually has a promoter, a single open reading frame encoding target library and following it in frame repA gene (that binds specifically to dsDNA). The template also has cis and ori sequences the RepA protein associates with.

The dsDNA template is then added to E. coli transcription-translation mixture (it could also be mixtures from other organisms). The RNA polymerase will produce mRNA from the template dsDNA and paused on CIS element. This is followed by the ribosome initiating translation on this mRNA-dsDNA complex. Newly translated (library peptide)-RepA fusion transiently associates with the CIS element on DNA template which eventually leads to RepA tightly binding adjacent ori sequence. Predominantly the nearest cis/ori region would be in the same DNA template that encodes cognate RepA-library fusion protein.

Obtained DNA-protein pool is incubated with an immobilized on a magnetic bead bait and nonbinding peptides got washed away while retained ones get eluted, PCR-amplified and usually use in the next round of selection.

Challenges

Apart from all the challenges that are normally associated with setting up an in vitro transcription-translation system, the CIS-display specifically is limited to the proteins that are (1) small and (2) well-fodlabe such that their function is not affected by the N-terminally fused RepA protein.

Future directions

The CIS display is used commerically and mainly is used in the search of single chan antibodies and peptide binders. The main disadvantage of CIS-display siilary to other in vitro protocols is that selection is done out of the cellular or orgamismal context. There is a report on utilising CIS-display in conjunction with in cellulo screening (https://pubs.acs.org/doi/10.1021/acschembio.7b00693) demonstrating the superious efficiency of such combined protocol.

Links to the protocols

https://link.springer.com/protocol/10.1007%2F978-1-0716-0720-6_10