# Targeting redundant gene families: A multiplexed, tissue-specific CRISPR toolbox for Arabidopsis genetic screens

## TL;DR

A new CRISPR toolbox for plants uses paired gene-targeting and barcoding to improve genetic screens, revealing hidden traits caused by redundant genes.

## Contribution

A multiplexed, tissue-specific CRISPR toolbox with double sgRNAs and barcoding to overcome genetic redundancy in plants.

## Key findings

- Multiplexed CRISPR libraries with two sgRNAs per construct improve gene coverage and editing efficiency.
- Barcoding enables tracking of sgRNA combinations in plants without sequencing each line individually.
- The toolbox was used to target 707 transporter genes in Arabidopsis, revealing hidden phenotypes due to redundancy.

## Abstract

Genome-scale targeted CRISPR libraries for forward genetic screens in plants are powerful tools for functional analysis, but they suffer from limited spatial control, single sgRNA design, and poor handling of genetic redundancy. We develop multiplexed CRISPR libraries in which each construct contains two sgRNAs that simultaneously target multiple members of a gene family. The libraries can also function at the cell-type-specific and tissue levels. A double-barcoding strategy enables efficient tracking and identification of sgRNA combinations at the plant level without individually sequencing each line. Using this platform, we generate over 1,000 Arabidopsis lines that express sgRNAs targeting 707 transporter genes across 114 gene families involved in nutrient uptake. The multiplexed design increases gene coverage and editing efficiency, underscoring its improved targeting capability to reveal hidden phenotypes. This toolbox provides a scalable resource for multi-targeted genome editing and spatially precise forward genetic screens in plants.

•Large multiplexed CRISPR libraries overcome redundancy, improving coverage and efficiency•A tissue-specific, multiplexed CRISPR library enables spatially precise editing in plants•Barcode-based “CRISPR-GuideMap” links sgRNAs to each plant, unmasks hidden phenotypes•SWEET13/14 transports trans-zeatin and functions redundantly with SWEET11/12

Large multiplexed CRISPR libraries overcome redundancy, improving coverage and efficiency

A tissue-specific, multiplexed CRISPR library enables spatially precise editing in plants

Barcode-based “CRISPR-GuideMap” links sgRNAs to each plant, unmasks hidden phenotypes

SWEET13/14 transports trans-zeatin and functions redundantly with SWEET11/12

Anfang et al. present a tissue-specific, multiplexed CRISPR toolbox for plants that uses paired sgRNAs and barcoded libraries to target redundant gene families. Applied to Arabidopsis nutrient transporters, it boosts editing efficiency and gene coverage, enabling spatially precise screens that uncover phenotypes hidden by functional redundancy.