Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Table 1 Oligonucleotides used in this study

Name	Sequence	Ref. No.
GS U6 R	AGGTTTGCTTCTCTTCGATTATG	1
TSO	GTCGCACGGTCCATCGCAGCAGTCACAGGGGG	2
U sense	GTCGCACGGTCCATCGCAGCAGTC	3
Fcp:Nat F	tggtctcaggagCTCGAGGTCGACGGTATC	4
Fcp:Nat R	aggtctcaagcgCGCAATTAACCCTCACTAAAGG	5
FCP prom F	tggtctcaggagAGCTTGCGCTTTTTCCGAG	6
FCP prom R	aggtctcacatTTTGGTATTGGTTTGGTAAATCAG	7
Cas9:YFP F	aggtctcaaATGGACAAGAAGTACTCCATTGG	8
Cas9:YFP R	aggtctcaaagcTCACTTGTACAGCTCGTCCATG	9
FCP term F	aggtctcagcttATACTGGATTGGTGAATCAATG	10
FCP term R	tggtctcaagcgGAGAACTGGAGCAGCTAC	11
U6 prom F	cggtctcaggagCTTCATCAAGAGAGCAACCA	12
U6 prom R	aggtctcaACAATTTCGGCAAAACGT	13
Urease sgRNA1 F	aggtctcattgt gtcgtaatcaagtattgccgGTTTTAGAGCTAGAAATAGCAAG	14
Urease sgRNA2 F	aggtctcattgt gtttccgatctaatgtccatGTTTTAGAGCTAGAAATAGCAAG	15
Urease sgRNA R	tggtctcaagcgTAATGCCAACTTTGTACAAG	16
FCP prom SDM F	TCCGCGGCAGaTCTCTGTCG	17
FCP prim SDM R	AGAAGTACCGTGTTGTTGCAGTG	18
NAT SDM F	CGACACCGTaTTCCGCGTCAC	19
NAT SDM R	GTGGTGAAGGACCCATCCAG	20
Cas9 screen F	CCGAGACAAGCAGAGTGGAAAG	21
Cas9 screen R	AGAGCCGATTGATGTCCAGTTC	22
NAT screen F	ATGACCACTCTTGACGACAC	23
NAT screen R	TTGATTCACCAATCCAGTATGC	24
Urease screen F 1	AAACAGACCACCTTCACCTC	25
Urease screen R	CTCCACCTGTACGTCTCG	26
Fcp seq F	CCATAAGTCAACGGCTCCAATC	27
NAT seq F	CTCTTGACGACACGGCTTAC	28
Cas9 seq 1 F	CATTACGGACGAGTACAAGGTG	29
Cas9 seq 2 F	TGAACACGGAGATCACCAAAG	30
Cas9 seq 3 F	CTTCCTGGACAATGAGGAGAAC	31
Cas9 seq 4 F	CAAACTGATCACACAACGGAAG	32
YFP FcpT seq F	ACTACCTGAGCTACCAGTCC	33
sg2 seq R	GTTTCCGATCTAATGTCCAT	34
sg1 seq F	TGTGTCGTAATCAAGTATTGC	35

Ref. No. 1–3: oligos used in 5′ RACE [35]. Ref. No. 4–16: primers for Golden Gate cloning, BsaI sites are underlined, 4 nt overhangs are shown in italics, and sgRNA targets are shown in bold. Upper case indicates complement to the template. Ref. No. 17–20: primers for SDM, lower case indicates base change. Ref. No. 21–26: primers for screening transformants. Ref. No. 27–35: primers for sequencing the CRISPR-Cas construct

ISSN: 1746-4811