# Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

## TL;DR

This paper introduces a new method for designing and predicting effective CRISPR/Cas9 tools in sea squirts to study gene functions in specific tissues.

## Contribution

The study introduces TuniCUT, a novel algorithm for predicting sgRNA efficacy, and provides a genome-wide sgRNA library for Ciona.

## Key findings

- Using two highly active sgRNAs, large genomic deletions can be achieved in Ciona embryos.
- TuniCUT algorithm predicts mutagenesis rates for 4.85 million potential sgRNA sites in the Ciona genome.
- A new OSO-PCR method allows rapid assembly of sgRNA cassettes for direct use in embryos.

## Abstract

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide. RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos.

We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.