Templated Repair: Depending on the extent of your desired genome edit, you can synthesize the repair template of single or double-stranded DNA.This strategy is used for simple gene disruptions. Untemplated Repair: For an un-templated edit, you do not require a repair template.Decide on what type of genome edit you want.These steps are based on using standard Streptococcus pyogenes Cas9 (SpCas9). The following steps outline what is required to perform CRISPR in a generic experimental system. These larger or more precise repair templates depend on Homology Directed Repair (HDR) to be introduced into the genome. If your goal is to insert larger or smaller gene fragments or introduce a very specific genetic change, then you would build a repair template that meets your needs. You may choose an un-templated genome edit if you want to simply disrupt the coding region of a gene. An untemplated repair event is achieved by Non-Homologous End Joining (NHEJ). The presence or absence of a repair template determines which repair mechanism is activated. In intact cells, DNA damage is immediately subject to repair, either un-templated DNA repair or templated DNA repair. Alternatively, you can design a single guide RNA, or sgRNA, which includes the gRNA sequence and tracrRNA sequence in one molecule. The gRNA and tracrRNA can be provided separately as described above.
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