Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays

Abstract

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

“…We hypothesized that the amounts of AcrIIA4-Cdt1 and SpyCas9 were strictly regulated, as in previous reports using T2A peptide 36 – 39 . Although a proline residue is added to the N-terminus of SpyCas9 after cleavage of T2A, SpyCas9 activity should not be affected by this, as the N-terminus of SpyCas9 is exposed to the surface in the apo form or in the complex with guide RNA and target DNA 40 . As the expression plasmid was changed to an episomal vector, the encoded proteins could be stably expressed without gene integration into the host genome.…”

Evolutionary simulations ofZ-linked suppression gene drives

“…This may not be necessary if the W-shredder targets many W-linked loci, but it is an important design consideration for the male gene drive component of the W-shredder, because the evolution of Z-linked resistance completely nullified the usefulness of male gene drive in the simulation (echoing [18]). Recent work demonstrated the feasibility of arrays containing many guide RNAs separated by spacers [38], suggesting it may soon be easier to create gene drives with multiple guide RNAs. A final way of developing resistance is to 'disarm' the endonuclease responsible for W-shredding and gene conversion, for example, by producing a protein that inhibits Cas9, or through evolution of gene regulatory processes that cause the Cas9 to no longer be expressed at the right time.…”

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

“…However, random insertion of multiple gRNA cassettes at a single locus may introduce transgene silencing (Lowder et al 2016;Vaucheret and Fagard 2001). gRNA expression cassettes can also be delivered through multiple DNA plasmids; however, this method is often associated with poor efficiency and cytotoxicity (Kurata et al 2018;Mans et al 2015;Peng et al 2016;Zhang and Matlashewski 2015). Moreover, each Agrobacterium strain only carries one type of T-DNA plasmid for transformation, making the delivery of multiple plasmids impractical in many organisms (Minkenberg et al 2017).…”

“…This system was applied to express multiple gRNAs or truncated gRNAs for dimeric CRISPR RNA-guided FokI nucleases (RFNs), and achieved higher editing efficiencies in mammalian system (Tsai et al 2014;Wyvekens et al 2015). Recently, a single Pol II promoter-driven gRNA array, which contains up to 10 gRNAs linked by optimal Csy4 ribonuclease sequences have been assembled by golden gate assembly for multiplex genome engineering in human cells (Kurata et al 2018). The Csy4-based system using a Pol II promoter, Cestrum Yellow Leaf Curling Virus promoter (CmYLCV), was also demonstrated to be successful for plant genome editing (Č ermák et al 2017).…”