Now Removing More Than 12,000bp! Knock-outs of genes are a commonly used tool for biologists to understand gene function 1 . Examination of phenotypes when the gene is deleted can reveal insights into what role that gene plays in the organism. We have found that a CRISPR approach using Cas9 complexes and short-homology arms 2 can be very effective at creating genomic deletions.
The following is an example of a 12.8kb deletion of all of the coding of the mod-5 locus. Using our proprietary algorithms, we selected two genome-targeting sgRNA sites flanking the genomic edit site. The first sgRNA targeted a sequence 100bp upstream from the ATG start codon. The second sgRNA was in the 3’ UTR at 100bp downstream from the TAG stop codon (Figure 1A). This deletion eliminated 100% of the mod-5 coding region. Our donor homology is a Oligo Deoxyribo-Nucleotide (ODN) designed to have 35bp homology arms with 55bp of content between the two arms (Figure 1B). The sgRNAs and ODN were ordered synthetically. We combined these with a co-CRISPR marker 3 (sgRNA and ODN) and Cas9 protein. Eighteen N2 animals were injected. Five days later, we screened for the phenotype caused by the insertion of the co-CRISPR marker mutation. Twenty-four F1 animals were then isolated and allowed to lay progeny. The adults were harvested for PCR to identify deletion mutants. Our three-primer PCR approach would only generate a band of 813bp if the deletion occurred and 614bp in wild-type (Figure 1C). We saw 3 lines that contained the deletion band. We sequenced the deletion and saw that repair had occurred correctly.
Figure 1. Modification of the mod-5 locus. A. Drawing representing the native mod-5 locus. Exons are blue arrows. sgRNAs used for the KO are in yellow. B. Drawing representing the donor homology ODN used for the repair to make the KO line. 35bp homology arms are shown in orange. C. Gel image showing the size difference between wild-type and the KO line. 1kb ladder was used with the band sizes 250bp, 500bp, 750bp, and 1kb shown.
Using this method, we have been able to identify deletion mutants from seven of seven client projects and three of four internal research projects (Table 1). The only failed project had a genomic region considerably larger than the others (31.2kb). Failure was likely due to a small screen size deployed for a very rare event. Only 20 to 35 F1 animals are typically screened, so it may be that screening more animals would allow us to isolate very large deletions, but it may be more effective to make a large deletion sequentially in small segments. On average, 46% of the F1s we screened contained a deletion. Intriguingly two projects had oligo-guided precision deletion in all of the the F1 co-CRISPR animals; however, the efficiency of deletion generation does not always directly correlate with deletion size (see Table 1). This may be an indication that sgRNA cutting efficiency is a larger determinant in biallelic deletions. Our average deletion size for the projects that worked is 5002.5bp. The largest deletion we have made is 12,775bp.
We are excited by these results and what it means for the C. elegans research community. Large genomic deletions can now become commonplace and will allow for scientist to gain a greater understanding of biology by being able to make complete loss-of-function alleles.
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- Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.
- Gagnon JA, Valen E, Thyme SB, et al. Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs. PLoS One. 2014; 9(5): e98186.
- Kim H, Ishidate T, Ghanta KS, et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics. 2014 Aug; 197(4):1069-80.