Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans. Issue 1 (15th November 2021)

• Record Type: Journal Article
• Authors: Medley, Jeffrey C; Hebbar, Shilpa; Sydzyik, Joel T; Zinovyeva, Anna Y
• Editors: Buelow, H
• Is Part Of: Genetics. Volume 220:Issue 1(2022)
• Publication Date: 2021-11-15
• Subjects: genome editing -- CRISPR -- Cas9 -- blocking -- scarless -- miRNA -- noncoding RNA -- C. elegans -- let-7; Genetics -- Periodicals; 576.5
• DOI: 10.1093/genetics/iyab199
• Languages: English
• ISSNs: 0016-6731

Abstract

Abstract: In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize "off-target" sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans . Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.