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Abstract Background The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. Results Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA. Conclusions CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to search variant landscapes based on ex vivo or in vivo mutagenesis, to identify and select new-to-nature and optimized properties in biomolecules. Yet, the majority of such systems rely on tedious iterations of library preparation, propagation, and selection steps. Furthermore, among the relatively few in vivo directed evolution systems developed to mitigate handling of repetitive ex vivo steps, directed evolution of DNA is the standard approach. Here, we present the protocol for designing the transfer of genetic information from evolving RNA donors to DNA in baker's yeast, using CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE). We use mutant T7 RNA polymerase to introduce mutations in RNA donors, while incorporation into DNA is directed by CRISPR/Cas9. As such, CRAIDE offers an opportunity to study fundamental questions, such as RNA's contribution to the evolution of DNA-based life on Earth. Graphic abstract: CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE)., Competing Interests: Competing interestsThe authors declare there are no potential conflicts of interest., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.