CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.

Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.

Boel A ，De Saffel H ，Steyaert W ，Callewaert B ，De Paepe A ，Coucke PJ ，Willaert A ... -  《-》

Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.

Richardson CD ，Ray GJ ，DeWitt MA ，Curie GL ，Corn JE ... -  《-》

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

DiNapoli SE ，Martinez-McFaline R ，Gribbin CK ，Wrighton PJ ，Balgobin CA ，Nelson I ，Leonard A ，Maskin CR ，Shwartz A ，Quenzer ED ，Mailhiot D ，Kao C ，McConnell SC ，de Jong JLO ，Goessling W ，Houvras Y ... -  《-》

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish.

Bai H ，Liu L ，An K ，Lu X ，Harrison M ，Zhao Y ，Yan R ，Lu Z ，Li S ，Lin S ，Liang F ，Qin W ... -  《BMC GENOMICS》

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

Paquet D ，Kwart D ，Chen A ，Sproul A ，Jacob S ，Teo S ，Olsen KM ，Gregg A ，Noggle S ，Tessier-Lavigne M ... -  《-》