Table 1.

Analysis of targeting vector knockin by CRISPR/Cas9 in mouse and rat zygotes.

Targeted Gene	Purpose 1	Cas9 Format 2	DNA Donor Format 3	Efficiency 4	Reference
Pitx1	Conditional	RNP	ssDNA	5.3	[108]
Ambra1	Conditional	RNP	ssDNA	9.5	[108]
Col12a1	Conditional	RNP	ssDNA	3.8	[108]
Ubr5	Conditional	RNP	ssDNA	12.5	[108]
Syt1	Conditional	RNP	ssDNA	2.2	[108]
Syt9	Conditional	RNP	ssDNA	2.4	[108]
PPP2r2a	Conditional	RNP	ssDNA	9.1	[108]
Fgf8	Reporter	RNP	ssDNA	7.7	[108]
Slc26a5	Reporter	RNP	ssDNA	4.5	[108]
Mafb	Reporter	RNP	ssDNA	3.8	[108]
Otoa	Reporter	RNP	ssDNA	5.6	[108]
Mmp9	Reporter	RNP	ssDNA	16.0	[108]
Mmp13	Reporter	RNP	ssDNA	7.7	[108]
Sox2	Reporter	Cas9 mRNA	dsDNA	2.0	[63]
Nanog	Reporter	Cas9-mSA	BioPCR	2.7	[63]
Gata6	Reporter	Cas9 mRNA	dsDNA	2.0	[63]
Gata6	Reporter	Cas9-mSA	BioPCR	5.0	[63]
Cdk9	Reporter	Cas9 mRNA	dsDNA	4.0	[63]
ROSA26	Reporter	Cas9 mRNA	dsDNA	1.3	[63]
Cdx2	Reporter	Cas9 mRNA	HMEJ	5.9	[110]
Cdx2	Reporter	Cas9 mRNA	Tild	1.9	[110]
Dbh	Reporter	Cas9 mRNA	Tild	3.6	[110]
Sp8	Reporter	Cas9 mRNA	HMEJ	3.2	[110]
Sp8	Reporter	Cas9 mRNA	Tild	2.0	[110]
Tdtomato	Reporter	Cas9 mRNA	Tild	3.5	[110]
Nr3c2	Conditional	Cas9 mRNA	Tild	4.8	[110]
Lhx6	Conditional	Cas9 mRNA	Tild	6.3	[110]
Serpina3	Conditional	Cas9 mRNA	ssDNA	3.5	[186]
Tyr	Conditional	Cas9 mRNA	ssDNA	2.0	[186]
mKIAA1322	Conditional	Cas9 mRNA	ssDNA	3.0	[186]
Serpina3n	Conditional	Cas9 mRNA	ssDNA	1.3	[186]
Mct4	Conditional	Cas9 mRNA	ssDNA	1.5	[186]
Rat Vapb	Conditional	Cas9 mRNA	ssDNA	3.9	[186]
ROSA26	Reporter	RNP	AAV	1.2	[187]
ROSA26	Reporter	RNP	AAV	4.8	[187]
Rat ROSA26	Reporter	RNP	AAV	4.2	[187]
Rat ROSA26	Reporter	RNP	AAV	5.4	[187]
ROSA26	Reporter	Cas9 mRNA	dsDNA	3.4	[112]
ROSA26	Reporter	Cas9 mRNA	dsDNA	2.1	[112]

¹ Conditional: A critical exon was flanked by loxP sites, so as to produce a Cre-dependent knockout allele. Reporter: an exogenous coding sequence, such as for a fluorescent protein, was inserted. ² RNP: ribonucleoprotein; Cas9 protein was complexed with guide RNA. Cas9 mRNA: in vitro transcribed mRNA from a plasmid containing Cas9 mixed with guide RNA. Cas9-mSa: in vitro transcribed mRNA from a plasmid containing Cas9 fused to monomeric streptavidin. ³ ssDNA: single-stranded DNA repair template. BioPCR: PCR was used to prepare biotinylated PCR amplicons. dsDNA: circular double-stranded DNA repair template. HMEJ: homology-mediated end joining; circular double-stranded DNA repair template incorporating sgRNA targets that flank homology arms. Tild: linear double-stranded DNA repair template. AAV: an adeno-associated vector donor was cultured with zygotes loaded with Cas9 RNP, by electroporation. ⁴ Efficiency, as calculated as the number of genetically engineered mice or rats produced per 100 zygotes treated with CRISPR/Cas9 reagents and transferred to pseudopregnant females.