These data, along with the large number of gRNAs required for the editing process, suggests that additional target specific factors are probably required for efficient editing.
While the initiating gRNA can begin the process by base pairing to the pre-edited mRNA, all subsequent gRNAs require editing of the target by the downstream gRNA to create the anchor-binding sequence necessary for recognition.
However, exclusive expression of TAP-tagged KREPA3ZFm2 resulted in accumulation of partially edited ATP synthase 6 mRNA that was more extensively edited and required the use of multiple gRNAs; similar results were obtained from cells exclusively expressing this mutant KREPA3 with myc-tag (Fig. 4 and Fig. S3).
When constructing binary vectors carrying multiple gRNAs, two or more PCR fragments are required.
Most pre-mRNAs require multiple cycles of RNA editing and multiple gRNAs to become functional.
However, to produce multiple gene mutations in plants, resources and methods for the assembly of multiple gRNA expression cassettes are frequently required.
Actually, we tried multiple gRNAs and still can not obtain cell clones with mutated polyA signal upstream of the distal pA site, possibly due to the inefficiency of the related gRNA (Fig. S15A).
To further improve the application of multiplex genome editing and to reduce the toxicity associated with multiple electroporation, we developed a One-shot CRISPR system, by incorporation of multiple gRNAs in a CAR lentiviral vector.
However, editing is a dynamic process that involves coordinated catalytic steps, and typically the use of multiple gRNAs [67].
More importantly, it can potentially target multiple genes in a single step because multiple gRNAs can share the same endonuclease Cas9 for multiple gene-KO [ 18, 27].
