Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9

Abstract

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Figure 1.

Design of knock-in strategies for tp53 and cdh5 point mutations and the AS-PCR assays for detection. (A) Genomic sequences and donor oligos are shown for sites of tp53 R143H, R217H and cdh5 G767S knock-ins. sgRNA sites are shown in dark blue, PAM sites and target codons are boxed and their mutations are highlighted in magenta and red, respectively. Mutations leading to introduction of BanI (boxed) and MspI (underlined) restriction sites are highlighted in light green. (B) Mutations induced by tp53 sgRNAs were detected by HMA using 10% PAGE analysis. Detection of indel mutations induced by cdh5 sgRNA was performed using the T7 Endonuclease I assay. Comparison of PCR product samples from uninjected zebrafish embryos with those injected with respective sgRNAs shows the degree of sgRNA effectiveness. (C) Restriction analysis of PCR products from uninjected, oligo-injected and tp53 knock-in injected embryos with enzymes introduced into donor oligos. (D) Allele-specific PCR assays for detecting introduced knock-ins are based on the idea that when two or more nucleotides are different between the WT and knock-in alleles, it is possible to design primers that can distinguish the two. Primers common to both WT and knock-in assays are highlighted in green and the site for variable primers is highlighted in gray. The WT and knock-in primers are indicated below or above the site and the nucleotides corresponding to the knock-in allele are in red both in the amplicon and the knock-in primer. (E) Example AS-PCRs are shown that were run on the extracts of pooled embryos from uninjected, oligo-injected, knock-in-injected samples and water. Both WT and knock-in AS-PCRs are shown, with the WT PCRs typically being very strong and indicative of the expected size for the correct knock-in AS-PCR products (indicated with red arrowheads).

Figure 2.

High-throughput sequencing analysis of point mutation knock-ins into the tp53 gene. (A) Quantification of the total fractions of small insertions and deletions in tp53 R143H and R217H knock-in injected embryo samples. The proportion of the reads with deletions or insertions represents a measure of sgRNA activity. (B) Measurements of R143H and R217H correct HDR knock-ins, knock-ins with additional insertions and deletions as well as knock-in reads with more aberrant complex events (unmapped). (C) Examples of different classes of R143H knock-ins: correct HDR knock-ins, knock-ins with deletions or insertions and unmapped knock-ins aligned to WT and expected knock-in sequences. (D) Examples of different classes of R217H knock-ins: correct HDR knock-ins, knock-ins with deletions or insertions and unmapped knock-ins aligned to WT and expected knock-in sequences.

Figure 3.

Enhancement of knock-in efficiency by anti-sense asymmetric oligos. Genomic sequences and donor oligos (sense symmetric and anti-sense asymmetric) are shown for sites of tp53 R143H (A) and R217H (B) knock-ins. All gel image panels show WT embryo genomic DNA extracts run with the tp53 R143H (C) or R217H (D) WT assays to serve as controls for PCR product sizes. As negative controls, knock-in (KI) assays for R143H (C) and R217H (D) were run on WT embryos or embryos injeсted with the respective oligos only. Knock-ins into tp53 to insert the R143H mutation were performed with either 126-nt sense symmetric oligo or with the 126-nt anti-sense asymmetric oligo (36 nt overlapping sgRNA site and 90 nt on the other side) (A and C). In the case of tp53 R217H, the sense symmetric oligo was 136-nt due to a larger distance from the cut site to the mutation and the anti-sense asymmetric one was 126-nt and had the same structure as the asymmetric R143H oligo (B and D). Samples of 16 individual embryos were taken from each knock-in injection and the knock-in assays performed on all of them under the same conditions and at the same time. The gel images shown are representative of at least three independent injections and corresponding comparisons of sense symmetric and anti-sense asymmetric oligo knock-in approaches. (E) Plot of total percentages of insertions, deletions and WT sequence reads at knock-in sites of tp53 R143H and R217H knock-in samples performed either with anti-sense asymmetric (anti) or sense symmetric (sense) ssODNs. (F) Quantification of percentages of different types of knock-ins at knock-in sites of tp53 R143H and R217H knock-in samples performed either with anti or sense ssODNs. Square root was applied to the percentage axis for the knock-in plot to better distinguish different values. Bars indicate the mean level for 3 replicates, whose values are plotted with green or orange circles for anti and sense oligos. The significance of differences in knock-in rates was determined using t-test and the P-value level is indicated above the respective items (**P-value < 0.01, *P-value < 0.05).

Figure 4.

AS-PCR-based strategy enables rapid screening and confirmation of potential founders and knock-in F1 embryos. (A) In the first step of this workflow, fish injected with the verified knock-in mix (Cas9, sgRNA and oligo) are grown to adulthood and then outcrossed to WT fish. The clutches derived from the breedings are used to prepare pooled genomic DNA samples from 50–100 embryos. WT and knock-in PCR assays are then run on these samples to identify the founders with detectable levels of germ-line transmission and to provide the size marker for knock-in AS-PCR products, which should be the same size as WT assay products. The information from the first screening round is then used to determine which founders should be bred or which available clutches should be chosen for subsequent screening of 24 individual embryos from each clutch. Knock-in assays are then performed on single-embryo samples and upon obtaining the results, positive embryo extracts are used to amplify a region of DNA surrounding the knock-in site and the resulting PCR products are sent for sequencing to determine if correct knock-in has actually occurred. (B andC) WT and knock-in AS-PCR are shown for cdh5 G767S (B) and tp53 R143H (C) knock-in screening of extracts from embryo clutches produced by potential founders. (D andE) Screening examples of 24 individual embryos from cdh5 G767S and tp53 R143H knock-in founders by AS-PCR. WT and knock-in AS-PCR on two WT embryo samples are shown as controls for PCR product size and as negative controls, respectively. (F) Sequencing chromatograms from WT and heterozygous cdh5 G767S F1 embryos and alignment of the corresponding sequencing reads confirm successful knock-in at the genomic level. (G) Sequencing chromatograms from WT and heterozygous tp53 R143H F1 embryos and alignment of the corresponding sequencing reads confirm successful knock-in at the genomic level.

Figure 5.

Knock-in analysis of cDNAs isolated from F1 carrier zebrafish. Analysis of cDNA cloned from F1 knock-in zebrafish with introduced tp53 R143H (A), tp53 R217H (B) or cdh5 G767S (C) mutations was performed by cloning cDNA fragments, identifying bacterial clones carrying either WT cDNA plasmids or those with knock-in mutations introduced, sequencing them and aligning the reads to the expected WT and knock-in cDNA sequences. Each of the alignments was performed with WT and mutant cDNA reference sequences, four WT cDNA clones, four knock-in cDNA clones and relevant exons. For the tp53 R143H and R217H knock-ins (A and B) we show the junction of the 5′ exon and target exon, knock-in region in the target exon and the junction between the target exon and 3′ exon. For the cdh5 G767S knock-in, we only show the target exon alignment because the mutation is at the end of the last exon and therefore unlikely to affect splicing (C).

Figure 6.

Analysis of true and off-target (trans) knock-ins at tp53 R143H site. (A) Definitions of knock-in types. In true-positive knock-ins, the targeting oligo modifies the intended locus without off-target insertions, whereas in the trans knock-ins, insertion of the oligo occurs at an off-target locus. (B) A model of how AS-PCR can produce PCR products in both true-positive and trans knock-in situations. In the case of a true-positive knock-in case, standard PCR successfully amplifies the expected PCR product. A possible mechanism in the trans knock-in case is presented here involving an abortive PCR product strands from the WT intended knock-in locus and the trans knock-in off-target locus. Since the oligo and WT locus share significant homology, it is conceivable that very short abortive PCR products from the two loci can pair up and become amplified to the full PCR product in the next cycle thus initiating the exponential cycle of amplification leading to large amounts of PCR product visible as an apparent knock-in band. Screening and sequencing verification of true-positive (C, E and G) and trans (D, F and H) knock-ins. (C and D) A set of 15 F1 embryos from positive founders were analysed using tp53 R143H knock-in AS-PCR. WT and positive control samples were run with both the WT and knock-in PCRs as controls for the size of the PCR product and specificity of the assay. (E and F) Knock-in site assay PCRs were run on samples from both founders and then either run undigested (upper panel) or digested with BanI enzyme (lower panel) to detect the BanI site expected to be introduced by correct tp53 R143H mutation knock-in. The samples previously identified as positive for knock-in are marked with ‘+’. (G and H) R143H site assay sequencing for true-positive and trans knock-in samples. Chromatograms show that in the true-positive knock-in base positions, there are double peaks (G), which are absent from the comparable trans knock-in read (H).

Figure 7.

PS-modified oligos improve knock-in consistency and efficiency. (A) Targeting scheme for introducing R471W knock-in into lamin A/C gene (lmna) in zebrafish using a sense asymmetric oligo. Red-colored lines indicate the donor oligo or DNA-derived from it and blue lines indicate genomic DNA. (B) Chemical structures of the PO and the PS groups show that one of the oxygens in PO is substituted with a sulphur atom in PS. The PS groups were added in the last two chemical bonds on either end of the PS-oligo for lmna R471W knock-in and the PO-oligo was synthesized in a standard way. (C) An example of gel data for AS-PCR analysis of lmna R471W knock-ins using PS and PO oligos. WT assay serves as a control for size of the products and the knock-in assay detects the modification. (D) Graph of measured intensities of AS-PCR signals for 44 embryos for each of the PO- and PS-oligo knock-in injected groups derived from three independent experiments. The data are aggregated because there was little variation between experiments. The type of oligo is indicated by color and with x-axis label. The ‘***’ indicate the P-value in t-test of < 0.001 (3.9e-07).