Strategies for genome editing using CRISPR/Cas9. a Gene knockout—CRISPR-enhanced HDR can be employed to replace a critical exon with a selectable drug resistance cassette (drug R), on one allele, relying on NHEJ-dependent indels to disrupt the other allele. b Gene knockout—A CRISPR-induced DSB can be used to efficiently introduce indels on both alleles. c Conditional knockout by inversion (COIN)—CRISPR-enhanced HDR can be used to introduce a Cre-recombinase invertible cassette, flanked by loxP sites (black triangles) into an artificial intron. This contains a splice acceptor site followed by a transcriptional termination signal (pA), so in one orientation it causes premature termination and mutation of the gene. In the opposite orientation, splicing occurs around the cassette, allowing the normal gene product to be produced from this allele. The second allele is disrupted by NHEJ-induced indels as in (a). d SNP introduction—A CRISPR-induced DSB is used to enhance HDR with a 100–200 nt ssDNA oligonucleotide repair template (green) to introduce small defined changes. e Scarless SNP introduction—A selectable marker cassette (drug R, green) is introduced into an intron or non-functional region along with the SNP of interest, and subsequently removed by a further round of HDR, or the piggyBac transposase. f Scarless SNP introduction—A SNP of interest is introduced as in D along with second site mutations necessary to prevent re-cleavage by the Cas9 enzyme. A subsequent second round of editing in a similar manner corrects the secondary mutations to leave only the SNP of interest. g Epigenetic editing—Catalytically dead Cas9 protein is used to recruit a variety of enzymatic activities (Enz, green circle) to specific sites, leading to transcriptional modulation (both positively and negatively), DNA or histone modifications such as DNA methylation, histone acetylation, methylation or phosphorylation, or cytosine deamination
