Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

Abstract

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Keywords: Cas9; Genome editing; Knockout; Mouse.

Fig. 1

Experimental parameters affecting Cas9-mediated mutant mouse line production. (a) Violin plot for founder rates from experiments with different methods of reagent delivery (CY, cytoplasmic injection; EP, electroporation; PN, pronuclear injection). Reported are the p-values (p) of pairwise comparisons using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (b) Violin plot for founder rates from experiments using two (2) or four (4) guide RNAs designed to produce deletion alleles. Reported is the p-value of pairwise comparison using the Wilcoxon rank sum test, the number of genes (N) and corresponding medians in boxplots. (c) Violin plot for founder rates from experiments with Cas9 guide RNAs designed to delete different sizes (in bp) of critical regions (genomic DNA). Each bin has ~ 640 unique gene deletion attempts. Reported is the p-value (p) of the overall comparison using the Kruskal–Wallis test. (d) Barplot showing the percentage of genes with GLT of the desired deletion allele after breeding one (1), two (2), three (3) or four or more (4 +) founders. Pairwise comparison of GLT rates using the Pearson chi-square test with Holm’s correction showed a significant difference only when breeding one founder was compared to breeding three or four founders (** p = 0.004 for each comparison, 1 vs. 3 and 1 vs. 4 +). No difference between 1 vs.2 or 3 vs. 4 + was observed. Unique gene attempts are the first attempt with GLT of the desired allele or the last of a set of unsuccessful attempts for each gene. See materials and methods for a complete description of data filtering. GLT, germline transmission.

Fig. 1

Experimental parameters affecting Cas9-mediated mutant mouse line production. (a) Violin plot for founder rates from experiments with different methods of reagent delivery (CY, cytoplasmic injection; EP, electroporation; PN, pronuclear injection). Reported are the p-values (p) of pairwise comparisons using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (b) Violin plot for founder rates from experiments using two (2) or four (4) guide RNAs designed to produce deletion alleles. Reported is the p-value of pairwise comparison using the Wilcoxon rank sum test, the number of genes (N) and corresponding medians in boxplots. (c) Violin plot for founder rates from experiments with Cas9 guide RNAs designed to delete different sizes (in bp) of critical regions (genomic DNA). Each bin has ~ 640 unique gene deletion attempts. Reported is the p-value (p) of the overall comparison using the Kruskal–Wallis test. (d) Barplot showing the percentage of genes with GLT of the desired deletion allele after breeding one (1), two (2), three (3) or four or more (4 +) founders. Pairwise comparison of GLT rates using the Pearson chi-square test with Holm’s correction showed a significant difference only when breeding one founder was compared to breeding three or four founders (** p = 0.004 for each comparison, 1 vs. 3 and 1 vs. 4 +). No difference between 1 vs.2 or 3 vs. 4 + was observed. Unique gene attempts are the first attempt with GLT of the desired allele or the last of a set of unsuccessful attempts for each gene. See materials and methods for a complete description of data filtering. GLT, germline transmission.

Fig. 2

Percentage of genes with GLT of the desired allele. Proportions represent genes with GLT of the desired deletion allele (successful), were abandoned with no additional attempts (aborted), or were repeated in subsequent experiments (repeated).

Fig. 3

Effects of biological variables on founder and GLT rates for null allele production. (a) Violin plot of founder rates of cellular non-essential and essential genes. Reported are the p-value (p) of the pairwise comparison using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (b) Violin plot for founder rates of homozygous lethal and non-lethal genes. Reported are the p-value (p) of the pairwise comparison using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (c) Stacked bar chart showing GLT of null alleles for essential and non-essential genes with multiple attempts to produce a null allele. (d) Logistic regression model showing the association of each variable with the success of the attempt to generate founders. An odds ratio below 1 is associated with a reduced probability of success, an odds ratio above 1 is associated with an improved probability of success, and an odds ratio of 1 is associated with no effect on success. Table 2 has the odds ratios and p-values for each variable, with and without essentiality in the model, that assess the significance of the difference of the estimate from zero. Supplementary Table 5 has the full model output. Each attempt represents a unique gene with the first attempt that successfully generated the desired allele or the last unsuccessful attempt for each gene used for analysis. See materials and methods for a complete description of data filtering.

Fig. 3

Effects of biological variables on founder and GLT rates for null allele production. (a) Violin plot of founder rates of cellular non-essential and essential genes. Reported are the p-value (p) of the pairwise comparison using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (b) Violin plot for founder rates of homozygous lethal and non-lethal genes. Reported are the p-value (p) of the pairwise comparison using the Wilcoxon rank sum test, the number of genes (N), and corresponding medians in boxplots. (c) Stacked bar chart showing GLT of null alleles for essential and non-essential genes with multiple attempts to produce a null allele. (d) Logistic regression model showing the association of each variable with the success of the attempt to generate founders. An odds ratio below 1 is associated with a reduced probability of success, an odds ratio above 1 is associated with an improved probability of success, and an odds ratio of 1 is associated with no effect on success. Table 2 has the odds ratios and p-values for each variable, with and without essentiality in the model, that assess the significance of the difference of the estimate from zero. Supplementary Table 5 has the full model output. Each attempt represents a unique gene with the first attempt that successfully generated the desired allele or the last unsuccessful attempt for each gene used for analysis. See materials and methods for a complete description of data filtering.

Fig. 4

Summary of reasons accounting for germline transmission failures with founders that failed to transmit a quality-controlled deletion allele to the N1 generation and thus failed to establish a knockout mouse line.