In Vivo CRISPR Multiplexing in Insects

Justin Overcash, Graduate Research Assistant, Entomology, Virginia Tech, MORE ABOUT THE AUTHOR

Port et al 2016 have posted in bioRχiv a manuscript describing a system and set of vectors for producing multiple CRISPR gRNAs from a single RNA polymerase II or III transcript in Drosophila, resources that will  enhance the CRISPR toolbox in Drosophila and beyond. The primary topics covered in this paper are the use of sgRNAs flanked by tRNAs under the control of a pol lll promoter (U6:3), the tissue specific utilization of tRNA flanked sgRNAs in a GAL4-UAS system and the use of Cpf1 (a Cas9 alternative).

Port et al shows that by flanking sgRNAs with tRNAs it is possible to create a single RNA transcript containing multiple sgRNAs. This method utilizes endogenous RNases to excise sgRNAs from a single RNA increasing the simplicity of generating multiplex constructs, and possibly more importantly allowing for the use of both pol ll and pol lll promoters.

To demonstrate the versatility of this technique, Port et al created a U6:3-t::gRNA (tRNA flanked sgRNAs) construct with four separate sgRNAs targeting the same gene as well as U6:3-t::gRNA construct targeting multiple genes. Amazingly, when a single gene was targeted they observed 100% biallelic disruption of the targeted gene in almost all cases. When targeting several genes, they discovered a mutation trend dependent upon the distance of the sgRNA from the U6:3 promoter with a 90% mutation rate generated by the sgRNA closest to the promoter, a 69% mutation rate generated by the sgRNAs that were the second and third closest, and 31% mutation rate generated by the fourth and furthest sgRNA.

tRNA Transcription

Following transcription of a cluster of tRNAs, each tRNA is clipped from the transcript. By locating these tRNA clipping sites on either side of an sgRNA on can use the endogenous tRNA transcript maturation process to produce functional guide RNAs in vivo.

Utilizing this technique, Port et al was also able to import their sgRNA expression method into the GAL4-UAS system. This was possible because the sgRNAs are flanked by tRNAs and excised in the nucleus, allowing for the use of a pol ll promoter  to drive sgRNA expression.

To demonstrate the advantage of utilizing t::gRNAs, Port et al compared the tissue specific expression of UAS-t::gRNAs to the ubiquitously expressed U6:3-gRNAs with both containing sgRNAs targeting the secretion factor wntless in the wing imaginal discs (Wls protein) via the posterior specific hedgehog promoter. The UAS-t::gRNAs outperformed the ubiquitously expressed U6:3-gRNA in both the removal of the Wls protein in the posterior compartment (no detectable Wls protein vs 60% removal for U6:3-gRNA) and in retaining tissue specificity to the posterior compartment (10% had small anterior cell patches with Wls protein reduction while 60% of U6:3-gRNA samples contained no or reduced Wls protein in cell patches that ranged from small to half of the anterior).

Lastly, Port et al tested the new Cas9 alternative Cpf1 which had thus far only been tested in mammalian cells.

The Cpf1 CRISPR-Cas protein expands genome-editing tools. Genome Biology201516:251

Schematic comparison of target recognition and degradation by Cpf1 and Cas9. An R-loop is formed as a result of protospacer adjacent motif (PAM) recognition (yellow), and subsequent base-pairing interactions occur between the CRISPR RNA (crRNA) and its cognate target sequence. Note that the guide RNA in Cas9 is an RNA duplex involving crRNA and trans-activating CRISPR RNA (tracrRNA), whereas Cpf1 uses a single crRNA. Upon sufficient complementarity in the seed region (red), Cpf1 and Cas9 nucleases will make two single-stranded cuts (blue triangles) resulting in a double-stranded break. DNA and crRNA lengths and cleavage positions are schematic only and are not drawn to scale. This figure and legend is from DOI: 10.1186/s13059-015-0824-9

The Cpf1 endonuclease differs from Cas9 in that it generates a staggered cut site which is further upstream of the PAM site than the blunt ended cut generated by Cas9. Cpf1 also has an alternative PAM which is T rich vs the G rich PAM site of Cas9.

Port et al tested sgRNAs with and without flanking tRNAs in combination with the Cpf1 endonuclease and found that the presence of tRNAs increased the mutation rate in founders and in offspring.

Yet, Port et al concluded that the whole of their observations suggest that Cpf1 may not be as effective in Drosophila as Cas9 and further improvements may be required before it becomes a competitive alternative.

Most of the technologies and resources described by Port et al. can be either used directly or easily adapted for use in non-drosophilid insect systems.

 

Port, F., Bullock, S. L., 2016 Expansion of the CRISPR toolbox in an animal with tRNA-flanked Cas9 and Cpf1 gRNAs. bioRχiv: doi: 10.1101/046417.

 

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