Efficient Knock-ins With Short Homology Arms

Andrew Paige is a Post-Baccalaureate Intramural Research Training Award Fellow in the Molecular Entomology Laboratory, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Washington, DC, United States where he is interested in using precise genetic technologies such as CRISPR-cas9 to create mutant knockouts of these genes with the goals of determining gene function and exploring their potential as candidates for transmission blocking vaccines. More About the Author

In a recent publication in eLIFE, Kanca et al (2019) describe three methods to create donors for efficient CRISPR-based insertion of homology donors in vitro and in vivo. Although pioneered in Drosophila, these methods may be useful for modifying other insect genomes.

Gene disruption is important for the functional genetic analysis of model and non-model organisms. The Bellen research group previously generated a multi-use transgenic library by inserting a Swappable Integration Cassette (SIC) into an intron flanked by two coding exons using transposon mediated integration or CRISPR-mediated homologous recombination, a technique called CRISPR-mediated Integration Cassette (CRIMIC). The CRIMIC SIC system creates loss of function alleles while expressing Gal4 with the pattern of the target gene. Gal4 can in turn drive transgenes with UAS regulatory sequences. This technique is powerful, but creating libraries of thousands of genes is resource intensive. The methods described in Kanca et al (2019) are intended to improve this system but will be widely useful. Like current knockin methods, these methods have pros and cons, which may make them more suitable for particular situations. Regardless, they are useful alternatives to consider for creating CRISPR knock-ins.

CRIMIC scheme. Coding intron sequence is cut by Cas9-gRNA complex. The sequences on either side of the cut site are used as 1-kb homology arms to integrate the SIC. Two possible SICs from published literature are included as examples. gRNA, guide RNA; RFP, red fluorescent protein; SIC, Swappable Insertion Cassette.

CRIMIC scheme. Coding intron sequence is cut by Cas9-gRNA complex. The sequences on either side of the cut site are used as 1-kb homology arms to integrate the SIC. Two possible SICs from published literature are included as examples. gRNA, guide RNA; RFP, red fluorescent protein; SIC, Swappable Insertion Cassette. Image

First Kanca et al (2019) developed a cloning free, PCR based method to produce 1 to 2 kb single stranded DNA donors. Using these ssDNA constructs, twelve of 20 target genes were established as GFP positive clones in S2R+ cells. These donors were less successful for targets with low expression levels, but they were twice as effective as traditional antibiotic selection marker methods. This ssDNA approach was successful in vivo, but it was less efficient than traditional dsDNA donors. However, the ease of production, cheap cost, and in vitro efficiency make it a great option to consider.

To improve integration efficiency of dsDNA donors, the authors induced in vivo linearization of plasmids using Cas9 express in germ cells and reduced the size of the Gal4 construct, the dominant marker, and the polyA sequence. A smaller dominant marker was used in the constructs, which is efficient but requires other transgenes for detection and segregates separately from the target gene. The minipolyA construct was less mutagenic, but the minipolyA-miniGal4 had results similar to CRIMIC. The decreased SIC size and in vivo linearization increased transgenesis rates, but the miniGal4 may be less successful for low expressed targets.

Image result for homology arms HDR

Homology arms of Donor DNA. Image

Finally, Kanca et al (2019) describe how the use of 100nt homology arms combined with in vivo linearization of the donor plasmid using Cas9 expressed in germ cells resulted in increased knockin rates. To reduce costs, the authors describe a modified cloning strategy to insert a commercially synthesized construct into a vector replacing the restriction cassette with the SIC of interest.
While motivated to improve genetic technologies designed for the functional genomic analysis of Drosophila melanogaster, the strategies for improving the efficiency of knockins and reducing the costs associated with creating donor molecules will be widely useful to insect biologists.

Kanca, O et (2019) An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms. Elife. 2019 Nov 1;8. pii: e51539. doi: 10.7554/eLife.51539.

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