**** New Dual positive (NPTII & Sl. pFAST) PLUS anti-selection (FCY-UPP) iCas9 (pICSL22022)/Cas12a (pICSL22023) acceptor constructs added. Both constructs contain an AmilRFP negative selection cassette and accept guides in all positions (1-7) via Bpi1. ****
Genome engineering is described as the as the precise manipulation of specific genomic sequences. It is performed using programmable molecular tools to create single-strand breaks (nicks) or double-strand breaks (DSBs) at specified locations in the genome and utilising endogenous mechanisms to repair the break. Depending what changes were made to the genome, genome engineering might be known as targeted integration (DNA was inserted at a specific genomic sequence), targeted mutagenesis (a small non-specific change such as an in-del to a specific genomic sequence) or gene editing (a specific change, or changes, of a specific genomic sequence e.g. recoding a gene). Non of these is the same as ‘classical’ or ‘first generation’ genetic modification (GM), which involves the random integration of new DNA into the genome.
There are a number of different tools that have been use to create double-stranded breaks:
You can learn more about using RNA-guided Cas9 endonuclease for engineering plant genomes HERE.
Most organisms have cellular mechanisms to repair damage to DNA induced by mutagens such as UV light and to resolve stalled replication forks formed during replication of genomic DNA. Once a break has been perceived, the cell will often arrest the cell-cycle to ensure that the error is not inherited by any daughter cells. Part cells will attempt to repair DSBs using any one of number of mechanisms including:
These endogenous mechanisms can be exploited and used to delete or modify endogenous DNA or to insert DNA at pre-determined sites.