Dataset Information

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

ABSTRACT: CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.

SUBMITTER: Zhao D

PROVIDER: S-EPMC9023296 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

Zhao Dongdong D Jiang Guo G Li Ju J Chen Xuxu X Li Siwei S Wang Jie J Zhou Zuping Z Pu Shiming S Dai Zhubo Z Ma Yanhe Y Bi Changhao C Zhang Xueli X

Nucleic acids research 20220401 7

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increase ...[more]

PMID: 35349689

Similar Datasets

Project description:The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5' and 3' ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5' tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCECandida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

Dataset Information

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

Publications

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets