SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish

Abstract

Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis.

Common strategies in silkworm disease resistance breeding research

“…Effective measures to prevent off‐target effects include the design of superior sgRNAs, selection of more stringent PAM sequences, and modification of Cas9 proteins. These approaches have been initially explored in plant and animal studies 78,79 . Thus, the silkworm could be modeled in a similar way to improve the application of CRISPR–Cas9 gene‐editing systems.…”

“…These approaches have been initially explored in plant and animal studies. 78,79 Thus, the silkworm could be modeled in a similar way to improve the application of CRISPR-Cas9 gene-editing systems. In addition, a method termed Improving Editing Activity by Synergistic Engineering (MIDAS) can simultaneously enhance the interaction between Cas proteins and PAM and ssDNA to improve the editing efficiency of the CRISPR-Cas system.…”

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

“…The use of Sp Cas9 variants free of PAM restrictions has emerged as a powerful option for DNA baseediting in both fundamental and applied research due to significantly improved target accessibility. Among the nucleases described thus far, an evolved Sp Cas9 nickase variant (termed SpRY) enabled nearly PAMless genome engineering in human cells [8], zebrafish [33], plants [34] and yeast [35]. In this work, SpRY was adopted as the basis to tailor a set of base-editors towards pushing the PAM-restriction boundaries in bacterial base-editing engineering.…”

Phenotype–genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution

“…com/#/) to visualize nucleotide sequences of the composite alleles and to calculate the rate of each allele and the total indel rate for each crispant (Conant et al, 2022). ICE analysis has been shown to produce genotyping results consistent with those obtained by nextgeneration sequencing analysis in previous crispant studies (Liang et al, 2022;Sieliwonczyk et al, 2023). We used the resulting ICE analysis output for further analysis only when the R 2 value indicating the reliability of the output was greater than the recommended value (0.8) (Conant et al, 2022).…”