- CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. - Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single- stranded DNA template to produce more efficient and precise mutations in zebrafish.. - zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr 25del/25del embryos, although more than half of the larvae had only a small number of pigmented cells. - Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. - Furthermore, quantification of zLOST knock-in efficiency at the rps14 , nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. - We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. - The CRISPR/Cas9 system has been efficiently used to achieve loss-of-function gene knockout in zebrafish with mutagenesis rates as high as . - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - However, such HDR-mediated knock-in approaches for genome editing have proved inefficient in zebrafish [9].. - It is possible to knock-in DNA sequences at specific loci through CRISPR/Cas9-mediated NHEJ in zebrafish, however NHEJ-based editing is not precise and the junc- tions between donors and break points are unpredictable [4, 10]. - Precise integration by HDR using a long double- stranded DNA donor (dsDNA) or single-stranded DNA oligonucleotides (ssODN) as homology repair templates has been achieved in zebrafish . - Anti-sense asymmetric oligo design was also found to be possible in zebrafish achieving around 2% efficiency of correct HDR knock-in as assessed by high-throughput sequencing analysis [14].. - Using a “base editing” (BE) system and zABE7.10 to induce point mutations in zebrafish in our lab, we were able to achieve base substitution at an efficiency between. - The template donors and target sites for HDR knock- in have varied widely, the latest iteration of which is a recently published method called Easi -CRISPR (Efficient additions with ssDNA inserts-CRISPR). - This strategy used long single- stranded DNA (lssDNA) donors with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes for two CRISPR-Cas9 sites at a single locus in order to generate correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live off- spring. - As such, this method can overcome the limita- tions of the other systems discussed above, but whether this strategy can be applied to the zebrafish model and how it compares to other DNA donors is still unknown.. - knock-in appeared to be enhanced by one order of mag- nitude from 5 to 98.5%. - To test whether the HDR se- quence modification was stably maintained in zebrafish somatic tissue, we used a restriction enzyme-based method and next generation sequencing (NGS) to test efficiency at three other sites ( rps14 , th and nop56. - Quantification of zLOST knock-in efficiency by next generation sequencing demonstrated that we achieved precise genome modification and its application resulted in over a dozen fold increased HDR efficiency in zebra- fish. - Overall, we demonstrate that zLOST pro- vides a simple and efficient method for inducing precise mutations in zebrafish.. - The tyr gene encodes tyrosinase which converts tyrosine into melanin, and a mutation in tyr results in an albino phenotype in zebrafish embryos;. - To ver- ify the reliability of the tyr 25del/25del mutant line, tyr. - Since melanophores of tyr 25del/25del. - are unable to produce melanin, this feature was used as a quick visible read-out for quantitatively comparing multiple repair template donors for HDR because of the correlation be- tween phenotypic rescue and knock-in efficiency.. - Comparison and optimisation of DNA template donors for HDR mediated knock-in efficiency. - For the circular dsDNA (cdsDNA) donor, the targeted genomic locus of tyr was amplified from wild type genomic DNA, and cloned into a pMD19-T vector both with and with- out two CRISPR target sites at both ends of the homolo- gous arms. - 1 CRISPR-mediated tyr knockout to establish a visual knock-in assay. - sequence, we directly injected zCas9 mRNA, tyr 25del/25del. - cdsDNA donors with two gRNA sites at both ends of the homologous arms (Fig. - To further validate the high HDR efficiency of zLOST, ssODN, and cdsDNA donors, we compared and quanti- fied the effectiveness of the tyr 25del/25del rescue assay. - For the cdsDNA donor, we found that only 5.4% of the lar- vae showed small numbers of pigmented cells at 2 dpf.. - For the ssODN donor with homologous arms, 39.1% of the larvae showed some cells with melanin production, a so-called “low rescue” or “medium rescue” phenotype.. - That may be because the low HDR effi- ciency observed with ssODN and cdsDNA occludes identification of the knock-in event by Sanger sequen- cing. - The efficiency of genome editing, regardless of the method used, is highly variable between different loci.. - Encouraged by the results of the tyr mutant gene rescue, we next investigated whether the relatively high effi- ciency of the zLOST method to precisely edit the zebra- fish genome was generally applicable to other loci. - Details of the target genes, lengths of the ssDNA repair templates, homology arms, and se- quencing data are shown in Fig. - However, we did not find “posi- tive embryos” using other knock-in strategies (data not shown). - T-A cloning of the zLOST-modified PCR prod- ucts followed by Sanger sequencing revealed that two out of 14 clones had seamless HDR modification, while three out of 14 clones carried indels (Fig. - It is worth noting that six out of the 14 clones sequenced showed incorrect-HDR knock-in, as they also showed deletions at the target site (KI + indels) (Fig. - 3c, where Δ1 and Δ2 are used to represent the indel, which is out of the shown sequence window). - Only two of the 21 adult fish that mated produced the desired Xho I identifiable allele and the germline transmitted mutations were confirmed by Sanger sequencing.. - 3b, top right) of which BamH I site conversion was observed in three of the 16 clones (Fig. - Taken together, these results demonstrate that knock-in zebrafish with specific point mutations can be generated with high efficiency using the zLOST strategy.. - Finally, we identified 4 nop56 founders ( n = 17) with tar- get knock-in mutations in their germline (23.5% germ- line transmission rate).. - However, none of these methods can truly assess the validity of HDR in depth because of the occultation of low frequency events. - Using Illumina sequencing restricted to the targeted region, we quantitatively com- pared the editing efficiency of the three strategies, ssODN, cdsDNA and zLOST. - How- ever, considering that random mutation could also occur in the vicinity of the gRNA site, we decided that random synonymous mutations, which do not change the encoded amino acid, would not preclude a sample being considered as a correct editing event. - Similar results were observed for the targeting of the th and rps14 loci (Fig. - b Restriction enzymes are used to digest the amplified region of the target genes. - c Sequencing results of the th, nop56 and rps14 loci. - 1 and △ 2 mean the presence of additional undesirable mutations outside of the shown sequence window.. - 4 NGS analysis of precise point mutation introduction to the genes th , nop56 and rps14 Total percentages of defined sequence reads classes at knock-in sites of th (a. - zLOST enables precise modelling of human disease mutations in zebrafish. - To this end, we tested the potential of zLOST to introduce human disease-related mutations in zebrafish. - Instead, we used zLOST to create a p.E78Q mutation in zebrafish (Fig. - Sequencing of the positive embryos successfully detected G to C con- version in 6 out of 15 clones (Fig. - We then went on to identify 7 founders ( n = 22) with a p.E78Q knock-in mutation in their germline (31.8% germline transmission rate).. - L51S) of the rpl18 gene is associated with DBA [19]. - However, these results provide a clear demonstration of the ability of zLOST to achieve HDR, and the utility of this to transmit precise knock-in alleles through the germline.. - HDR-mediated knock-in is a valuable approach for dis- ease modelling and functional analysis. - Although the applica- tion of lssDNA donors as a robust method for mouse genome editing has been previously reported [16], a broad application of HDR using lssDNA donors to many loci has yet to be achieved in zebrafish. - third, we found that zLOST produced the highest efficiency of the three strategies for HDR on multiple loci. - Antisense asymmetric oligo design was also found to be a success- ful optimised strategy in zebrafish [14]. - activity of the NHEJ pathway or boosting the activity of the HDR pathway. - germline transmission of point mutations up to 25% in zebrafish [24]. - 5 zLOST enables mimicking of human disease related mutations in zebrafishAlignment of human patients and desired zebrafish mutations to model human Barber-Say syndrome (BSS) or Diamond-Blackfan anaemia (DBA), schematic outlines of the gene editing strategy and sequencing of the resulting twist2 and rpl18 zebrafish loci. - a Diagram of the mutation associated with human BSS. - b and d Design principles of HDR templates that contain a non-synonymous mutation of the sequence close to the PAM site in addition to synonymous nucleotide changes that create a Coding-bar used for genotyping that utilizes a de novo endonuclease restriction site. - By comparing multiple donors, we conclude that zLOST can introduce accurate mutations to mimic human disease in zebrafish more efficiently, and as such can expedite the study of disease mechanisms and development of therapeutics.. - The PAM sites or seed sequences in the HDR donors were altered to prevent re-cutting of the desired donor DNA.. - Illumina-based sequencing to quantify knock-in rates (next generation sequencing). - To avoid interference by the residual donor DNA, we designed a pair of primers that are located outside the region of the donor DNA for the first round of PCR, which was purified to continue as the second round template for amplification. - It is also possible to use the linker sequence contained in the trim data of the quality control software. - After assembling, each read represents a sam- ple because the gene sample for sequencing is only 250 bp length, which is shorter than the length of the un- paired single reads of Illumina. - The correct editing event that meets our expectations is, within the range of gRNA, only our desired base sub- stitutions occurred without other substitutions or indel events and the protein translated from the cds of the gene only has the expected residue substitution after editing. - Of the region covered by gRNA of the gene, we refer to the reference sequence as WT_sgRNA_pattern, the sequence after correct editing as HDR_sgRNA_pat- tern. - When the HDR_sgRNA_pattern matched to the sample sequence, we checked to see if the protein sequence coded by the CDS region of the gene only contained expected residue substitutions. - qPCR analysis of the tyr expression in tyr 25del/25del zebrafish. - Sequencing result of tyr 25del/25del gRNA induced indels.. - KI: Knock in;. - HPB was responsible for the design of the experiments and manuscript writing. - HPB established the tyr 25del/25del. - The salary of the authors and the cost of experiments are supported in part by these funding resources: National Natural Science Foundation of China and Technology Program of Shenzhen (JCYJ . - These funding bodies were not directly involved in any part of the design of study, the collection, analysis, or interpretation of the data, or writing of the manuscript.. - All datasets supporting the conclusions of the manuscript are included within the article and its additional files. - Efficient genome editing in zebrafish using a CRISPR-Cas system. - Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. - Expansion of CRISPR/Cas9 genome targeting sites in zebrafish by Csy4-based RNA processing. - Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. - Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. - TALEN-mediated precise genome modification by homologous recombination in zebrafish. - Intron targeting-mediated and endogenous gene integrity-maintaining knockin in zebrafish using the CRISPR/Cas9 system. - Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. - Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. - Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. - Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. - RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. - Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. - Stages of embryonic development of the zebrafish
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt