Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta

Plant Molecular Biology

Ross A. Johnson1,*, Vyacheslav Gurevich1, Shdema Filler1, Aviva Samach1, and Avraham A. Levy1

1 Present address: Department of Plant Sciences, the Weizmann Institute of Science, Rehovot, Israel

* To whom correspondence should be addressed. Email:

Online Resource 6 Features of CRISPR-Cas sgRNAs relative to their cleavage performance, for our work and reports of others. Data for the plant and tissue used to test sgRNAs, the sgRNA sequence, their performance, and reference. All cleavage efficiency data shown from the work that we report here has been combined together from two separate experiments, and standard error (SE) values referring to differences between replicate experiments here, rather than by using a mega analysis of two experiments. Only previously reported studies with sgRNAs containing 20-nucleotide target binding regions were analyzed due to the same criteria being used by Wang et al. (2014). Previously reported studies are compared in terms of their targeted mutagenesis frequencies, which are a percentage. Sequences highlighted in light blue represent indicators of good sgRNA performance (namely a G, G/C, G, and G/A in the last 4 positions of the sgRNA’s 20 nucleotide target binding sequence, as well as an A in positions 4, 11, 12, 15, and 16 of this sequence), whereas sequences highlighted in red represent indicators of poor sgRNA performance (a U in the last 4 positions of the sgRNA’s target binding sequence). a Determined by delivering the sgRNA with the Cas9 construct. b Determined by delivering the sgRNA with the LUC-based reporter construct. c Determined by the number of plants with a single allelic mutation divided by the number of genotyped T1 transgenic plants. d Frequency of mutation in T2 plants. e Frequency of mutation in T0 plants.

Plant / Tissue / Sequence (5′ - 3′) / Sum of Performance Indicators / sgRNA performance / sgRNA name / Ref.
N. benthamiana / Agro-Infiltrated leaves / GAGCGTATATAATGCTGCTT / 2 / 120b –fold cleavage efficiency
(SE= 58, n=2) / PSY #2 / (This study)
GAATGTCTGTTGCCTTGTTA / 0 / 87b –fold (SE= 13, n=2) / PSY #4 / (This study)
GGGACAACAAGGCCAACCAT / 3 / 79a –fold (SE= 8.6, n=2) / T494 #2 / (This study)
GACAACAACAAGGCCAACCA / 5 / 76a –fold (SE= 5.3, n=8) / T494 #1 / (This study)
GCGATGCTACCAGCATTCTG / 2 / 58b –fold (SE= 19, n=2) / CrtISO #3 / (This study)
GTCCCTGTCCTTCCCATGGT / 1 / 30–fold
(SE= 0.13, n=2) / 3m / (This study)
GATATTCTTCCCCTCCTGAG / 2 / 32ab–fold (SE= 8.9, n=6) / Tqqr #3 / (This study)
GAGGCGTTCTTCCCCTCAGG / 2 / 18a,b –fold (SE= 2.7, n=6) / Tqqr #1 / (This study)
GTTGATGAGTGTTCCAATTA / 0 / 18a,b–fold (SE= 0.81, n=4) / PSY #1 / (This study)
GGAATTGTTTTAGACAACCA / 5 / 15a –fold (SE= 3.2, n=2) / PSY #5 / (This study)
GCTGGTAGATCTTCTAATAG / 1 / 10a–fold (SE= 6.5, n=2) / T852 / (This study)
GTGCTTTTACATAAGCTTTC / -2 / 2.2a –fold (SE= 0.40, n=2) / CrtISO #5 / (This study)
GCCGTTAATTTGAGAGTCCA / 2 / 2.1% targeted mutagenesis / NbPDS / (Nekrasov et al., 2013)
4.8% / NbPDS # 1 / (Li et al., 2013)
PEG-transfected leaf protoplasts / 37.7% / NbPDS # 1 / (Li et al., 2013)
GTCAAGATGTTTGCTTGCAA / 4 / 38.5% / NbPDS # 2 / (Li et al., 2013)
A. thaliana / PEG-transfected leaf protoplasts / GGACTTTTGCCAGCCATGGT / 2 / 5.6% / AtPDS3 / (Li et al., 2013)
GTTGCTCACGTAAGCGATTT / -2 / 1.1% / AtFLS2 / (Li et al., 2013)
Transgenic plants / TTGGGTCATAACGATATCTC / 1 / 84.2%c / BRI1 #1 / (Feng et al., 2013)
GCAATAGGAAGTTCTGTCAA / 2 / 81.7%c / JAZ1 #1 / (Feng et al., 2013)
GATGAGCTTCTAGCTGTTCT / -2 / 47.0%c / GAI #1 / (Feng et al., 2013)
TGACATACATGAGCTCCTGA / 2 / 33.3%c / BRI1 #2 / (Feng et al., 2013)
GTAAGAGCTGACATAGCCTG / 4 / 30.0%c / BRI1 #3 / (Feng et al., 2013)
ACCTCCGGCGTCGACATGCC / 1 / 28.3%d / TT4 / (Fauser et al., 2014)
CGTATCTTCGGCCATGAAGC / 2 / 13.8%d / ADH1 / (Fauser et al., 2014)
O. sativa / Transgenic plants / GCGTCGGCGGAGACAGAGCG / 3 / 91.6%c / LAZY1 / (Miao et al., 2013)
ACCGGACCACCTCAAGAGCT / 1 / 83.3%c / CAO1 / (Miao et al., 2013)
GCGCGCCACCTCGGCCGAAG / 2 / 75.0%c / YSA #1 / (Feng et al., 2013)
GGCGCGCGTCGAGGCGTTGG / 1 / 66.7% e / OsMYB1 / (Zhang et al., 2014)
GCGCGCCACCTCGGCCGAAG / 2 / 66.7% e / OsYSA / (Zhang et al., 2014)
GCGGAGAACGACAGCCGGTC / 2 / 65.1% e / OsROC5 / (Zhang et al., 2014)
CCGCGACGAGCACCTTCATG / 1 / 51.4% e / OsYSA / (Zhang et al., 2014)
GGAATTAGCGGCGGCGGCGA / 5 / 50.9% e / OsDERF1 / (Zhang et al., 2014)
GGCGATTGGGAGCCTCGGCG / 4 / 47.6%c / SPP #1 / (Feng et al., 2013)
CCGCGACGAGCACCTTCATG / 1 / 47.6%c / YSA #2 / (Feng et al., 2013)
GGAAGGATGAAGATGGAGAT / 2 / 41.9% e / OsPDS / (Zhang et al., 2014)
GGAGGTCCTGGTCTAAACCA / 4 / 37.0% e / OsMSH1 / (Zhang et al., 2014)
GTAGCCCTGCTGTTGAAGAA / 2 / 31.9% e / OsMYB5 / (Zhang et al., 2014)
GGCGATTGGGAGCCTCGGCG / 4 / 28.9% e / OsSPP / (Zhang et al., 2014)
CACCTACATATAGGAGTGGG / 4 / 26.3% e / OsPMS3 / (Zhang et al., 2014)
GCGGAGAACGACAGCCGGTC / 2 / 25.8%c / ROC5 #1 / (Feng et al., 2013)
GGATGTTCACTACATGCTTG / 0 / 21.1% e / OsEPSPS / (Zhang et al., 2014)