Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Abstract

Background: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations.

Results: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward.

Conclusions: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.

Keywords: CRISPR; Cas9; Embryo; Genome editing; Germline; Mutagenesis; Next generation sequencing; Perl; Zebrafish.

Fig. 1

CRISPR sgRNA design process. An Ensembl gene model is shown at the top; two exons are expanded in the rest of the diagram. The target sequence is searched for CRISPR sites, these are scored and the best scoring are selected. For screening, PCR primers are designed for making amplicons into sequencing-ready libraries. The package also includes a database schema for storing information on CRISPR designs and screening information

Fig. 2

CRISPR workflow. a Overall workflow. Diagram showing the steps of the process. b Strategy for sgRNA screening. Initially, sgRNAs are screened for efficiency and those with high cutting efficiency are re-injected. The G0 embryos are raised and males are screened for germline CRISPR-induced indels. For high-transmitting samples, embryos are generated by IVF, raised and the resulting F1 carriers are identified by KASP

Fig. 3

Crispr analysis pipeline. a Analysis pipeline. Schematic of sequence analysis procedure. b Pipeline outputs. Examples of the visualisations that the pipeline produces. Top—plate map showing the percentage of reads containing an indel for each sample along with the total number of reads. Bottom—display of induced variants relative to the CRISPR target site

Fig. 4

Comparison of induced indels between germline and somatic tissues. a Plot of total percentage of reads with an indel in sperm versus fin clip. b Plot of frequencies for individual variants in sperm versus fin clip. Variants that are present in both the sperm and fin-clip samples from a single individual are in red. Plot has been cropped to make points near the origin easier to distinguish. Figure S2 is the original. c Plot showing the average overlap of indels in each tissue for each sgRNA. d Plot showing the correlation between the frequency of reads from MiSeq data and the number of carriers identified in F1 outcrosses

Fig. 5

Efficiency of longer sgRNAs. a Longer sgRNAs (GGN21GG) tend to be less efficient than those designed to the usual consensus (GGN19GG). Plot shows the distribution of the mean indel frequency for sgRNAs with different design strategies. b Plot showing the distribution of induced indel frequencies for individual sgRNAs with different design strategies