PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants

Sebastian M Siegner,Zacharias Kontarakis,Jacob E Corn,Mehmet E Karasu,Markus S Schröder

doi:10.1186/s12859-021-04034-6

Abstract

BackgroundThe rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents.ResultsWe developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ConclusionWith PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.

Highlights

The rapid expansion of the Clustered regularly interspaced short palindromic repeats (CRISPR) toolbox through tagging effector domains to either enzymatically inactive CRISPR associated 9 (Cas9) or Cas9 nickase has led to several promising new gene editing strategies
Prime editing with PnB Designer user interface Selecting the ‘Prime editing’ option, the user can install a desired edit or correct a certain mutation of any type by setting the switch button to the left or right position (Fig. 1c)
While it is suggested to start with a primer binding site (PBS) length of ~ 13 nt and reverse transcriptase template (RTT) length of 10–16 nt, precise rules for their values have not yet been determined and prime editing extended guide RNA (pegRNA) efficiency can be optimized by varying RTT length [16]

Summary

Introduction

The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas (dCas9) or Cas nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. Fusing various effector domains to catalytically impaired versions of Cas proteins has led to an explosion of tools with outcomes beyond indels and HDR [2]. Adenine-deaminase base editors (ABEs) in particular exhibit a promising combination of a high efficiency at the on-target site with low off-target DNA and RNA editing [12, 13]. Some of the early CBEs versions suffered from off-target activity [12], and only the newly engineered versions of these CBEs or Target-AID based CBEs exhibit sufficient specificity to be considered for therapeutic applications [13,14,15]

Methods

Results

Conclusion