Table 2.

Optimization of short guide RNA targeting murine Drd2 gene by Cel-I assay and off-target prediction in silico

No.	Primers	Sequence	Strand	Domain	PAM	Cleavage efficiency (%)	Off target score (predicted)
1	Forward	5′-CACCGAAGATGTCACAGTGAATCCTG-3′	–	TM3	CTGAAT	0.0	65.9
	Reverse	5′-AAACCAGGATTCACTGTGACATCTTC-3′
2	Forward	5′-CACCGCGGTGCAGAGTTTCATGTCCT-3′	–	IC3	CAGGGT	0.0	74.4
	Reverse	5′-AAACAGGACATGAAACTCTGCACCGC-3′
3	Forward	5′-CACCGTTAGACTTCATGATAACGGTG-3′	–	IC3	CAGAGT	NA	91.2
	Reverse	5′-AAACCACCGTTATCATGAAGTCTAAC-3′
4	Forward	5′-CACCGACCGTTATCATGAAGTCTAAT-3′	+	IC3	GGGAGT	NA	89.1
	Reverse	5′-AAACATTAGACTTCATGATAACGGTC-3′
5	Forward	5′-CACCGGAGTTTCCCAGTGAACAGGCG-3′	+	IC3	GAGAAT	5.1	84.6
	Reverse	5′-AAACCGCCTGTTCACTGGGAAACTCC-3′
6	Forward	5′-CACCGCATCGTACCAGGACAGGTTCA-3′	–	N-term	GTGGAT	3.7	88.8
	Reverse	5′-AAACTGAACCTGTCCTGGTACGATGC-3′
7	Forward	5′-CACCGAGAACTGGAGCCGGCCCTTCA-3′	+	N-term	ATGGGT	0.0	84.0
	Reverse	5′-AAACTGAAGGGCCGGCTCCAGTTCTC-3′
8	Forward	5′-CACCGAAAGACGATGATAAAGATGAG-3′	–	TM1	GAGGGT	9.5	49.9
	Reverse	5′-AAACCTCATCTTTATCATCGTCTTTC-3′
9	Forward	5′-CACCGGGTGGCCACACTGGTTATGCC-3′	+	TM2	CTGGGT	11.0	88.6
	Reverse	5′-AAACGGCATAACCAGTGTGGCCACCC-3′
10	Forward	5′-CACCGCACACGCTACAGCTCCAAGCG-3′	+	IC2	CCGAGT	0.0	87.8
	Reverse	5′-AAACCGCTTGGAGCTGTAGCGTGTGC-3′
11	Forward	5′-CACCGTACTGTCATGATCGCCATTGT-3′	+	TM4	CTGGGT	7.2	85.0
	Reverse	5′-AAACACAATGGCGATCATGACAGTAC-3′