Fig. 7. Genomic integration with short homology arms.

(a) A single-stranded DNA donor (long ssODN) containing homology arms 30-100 bases (blue, green) is administered together with the CRISPR guide RNA and Cas9 which cuts the desired target site in the genome. Alternatively (not shown) the donor can be dsDNA with two short ssODNs providing the bridging homology arms. (b) For future conditional knockouts (as by Cre-recombinase in mice), two guide RNAs and two short ssODNs (each with a LoxP site) are introduced to result in LoxP sites (triangles) at the genomic sites where Cas9 cleavage had occurred. The LoxP sites allow future deletion for conditional knock-out. (c) A more efficient strategy for future conditional knock-outs is to use a single long ssODN donor with LoxP sites (triangles) flanking the homology arms at the ends (blue, green). As in panel (b), the genomic DNA is cut by Cas9 that is targeted by two guide RNAs. Note that cutting the genome with two gRNAs (panel c) can also be employed for DNA replacement in the genome, in contrast to a simple DNA insertion after cutting the genome with just one gRNA (panel a). Easi-CRISPR employs the method shown here in panel (c): A long single-stranded DNA donor with short homology arms is injected with the pre-assembled Cas9 ribonucleoprotein (ctRNP) complex containing two guide RNAs to create targeted insertion. The ctRNP:complex contains crRNA + tracRNA +Cas9 protein). The dashed line indicates the inserted DNA sequence in the single-stranded donor DNA and in the double-stranded recombinant DNA. The sequence indicated by the dashed line can be deleted readily in future experiments by use of the LoxP sites that flank it. (d) The “two-hit by sgRNA and two oligos with a targeting plasmid” (2H2OP) portrayed here introduces a double-stranded donor plasmid DNA and two short bridging ssODNs (providing homology to the junctions) together with two sgRNAs to direct Cas9 cleavage of the chromosomal target site and of the donor plasmid, respectively.