mediated gene knockout efficiency. Sci Rep
6:28566.
https://doi.org/10.1038/
srep28566
14. Sugano SS, Nishihama R, Shirakawa M et al
(2018) Efficient CRISPR/Cas9-based genome
editing and its application to conditional
genetic analysis in Marchantia polymorpha.
PLoS One 13(10):e0205117
15. Peterson BA, Haak DC, Nishimura MT et al
(2016) Genome-wide assessment of efficiency
and specificity in CRISPR/Cas9 mediated
multiple site targeting in Arabidopsis. PLoS
One 11(9):e0162169. https://doi.org/10.
1371/journal.pone.0162169
16. Feng C, Su H, Bai H et al (2018) Highefficiency genome editing using a dmc1
promoter-controlled CRISPR/Cas9 system in
maize. Plant Biotechnol J 16(11):1848–1857.
https://doi.org/10.1111/pbi.12920
17. Lee K, Zhang Y, Kleinstiver BP et al (2019)
Activities and specificities of CRISPR/Cas9
and Cas12a nucleases for targeted mutagenesis
in maize. Plant Biotechnol J 17(2):362–372.
https://doi.org/10.1111/pbi.12982
18. Tang X, Liu G, Zhou J et al (2018) A largescale whole-genome sequencing analysis
reveals highly specific genome editing by both
Cas9 and Cpf1 (Cas12a) nucleases in rice.
Genome Biol 19(1):84. https://doi.org/10.
1186/s13059-018-1458-5
19. Zhang Q, Xing H-L, Wang Z-P et al (2018)
Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis
and its prevention. Plant Mol Biol 96
(4-5):445–456.
https://doi.org/10.1007/
s11103-018-0709-x
20. Doench JG, Hartenian E, Graham DB et al
(2014) Rational design of highly active
sgRNAs for CRISPR-Cas9-mediated gene
inactivation.
Nat
Biotechnol
32
(12):1262–1267. https://doi.org/10.1038/
nbt.3026
21. Wang T, Wei JJ, Sabatini DM et al (2014)
Genetic screens in human cells using the
CRISPR-Cas9
system.
Science
343
(6166):80–84. https://doi.org/10.1126/sci
ence.1246981
22. Liang G, Zhang H, Lou D et al (2016) Selection of highly efficient sgRNAs for CRISPR/
Cas9-based plant genome editing. Sci Rep
6:21451.
https://doi.org/10.1038/
srep21451
23. Liu X, Homma A, Sayadi J et al (2016)
Sequence features associated with the cleavage
efficiency of CRISPR/Cas9 system. Sci Rep
6:19675.
https://doi.org/10.1038/
srep19675
24. Ma X, Zhang Q, Zhu Q et al (2015) A robust
CRISPR/Cas9 system for convenient, highefficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8
(8):1274–1284. https://doi.org/10.1016/j.
molp.2015.04.007
25. Xu H, Xiao T, Chen C-H et al (2015)
Sequence determinants of improved CRISPR
sgRNA
design.
Genome
Res
25
(8):1147–1157. https://doi.org/10.1101/gr.
191452.115
26. Le C, Ran FA, Cox D et al (2013) Multiplex
genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://
doi.org/10.1126/science.1231143
27. Zheng T, Hou Y, Zhang P et al (2017)
Profiling single-guide RNA specificity reveals
a mismatch sensitive core sequence. Sci Rep
7:40638.
https://doi.org/10.1038/
srep40638
28. Nishimasu H, Ran FA, Hsu PD et al (2014)
Crystal structure of Cas9 in complex with
guide RNA and target DNA. Cell 156
(5):935–949. https://doi.org/10.1016/j.cell.
2014.02.001
29. Konermann S, Brigham MD, Trevino AE et al
(2015) Genome-scale transcriptional activation
by an engineered CRISPR-Cas9 complex.
Nature 517(7536):583–588. https://doi.
org/10.1038/nature14136
30. Sander JD, Dahlborg EJ, Goodwin MJ et al
(2011) Selection-free zinc-finger-nuclease
engineering by context-dependent assembly
(CoDA). Nat Methods 8(1):67–69. https://
doi.org/10.1038/nmeth.1542
31. Sanjana NE, Le C, Zhou Y et al (2012) A
transcription activator-like effector toolbox
for genome engineering. Nat Protoc 7
(1):171–192.
https://doi.org/10.1038/
nprot.2011.431
32. Verkuijl SA, Rots MG (2019) The influence of
eukaryotic chromatin state on CRISPR-Cas9
editing efficiencies. Curr Opin Biotechnol
55:68–73.
https://doi.org/10.1016/j.
copbio.2018.07.005
33. Horlbeck MA, Witkowsky LB, Guglielmi B
et al (2016) Nucleosomes impede Cas9 access
to DNA in vivo and in vitro. elife 5:e12677.
https://doi.org/10.7554/eLife.12677
34. Wu X, Scott DA, Kriz AJ et al (2014) Genomewide binding of the CRISPR endonuclease
Cas9 in mammalian cells. Nat Biotechnol 32
(7):670–676. https://doi.org/10.1038/nbt.
2889
35. Daer RM, Cutts JP, Brafman DA et al (2017)
The impact of chromatin dynamics on Cas9mediated genome editing in human cells. ACS
Guidelines for gRNA Design
341
6:28566.
https://doi.org/10.1038/
srep28566
14. Sugano SS, Nishihama R, Shirakawa M et al
(2018) Efficient CRISPR/Cas9-based genome
editing and its application to conditional
genetic analysis in Marchantia polymorpha.
PLoS One 13(10):e0205117
15. Peterson BA, Haak DC, Nishimura MT et al
(2016) Genome-wide assessment of efficiency
and specificity in CRISPR/Cas9 mediated
multiple site targeting in Arabidopsis. PLoS
One 11(9):e0162169. https://doi.org/10.
1371/journal.pone.0162169
16. Feng C, Su H, Bai H et al (2018) Highefficiency genome editing using a dmc1
promoter-controlled CRISPR/Cas9 system in
maize. Plant Biotechnol J 16(11):1848–1857.
https://doi.org/10.1111/pbi.12920
17. Lee K, Zhang Y, Kleinstiver BP et al (2019)
Activities and specificities of CRISPR/Cas9
and Cas12a nucleases for targeted mutagenesis
in maize. Plant Biotechnol J 17(2):362–372.
https://doi.org/10.1111/pbi.12982
18. Tang X, Liu G, Zhou J et al (2018) A largescale whole-genome sequencing analysis
reveals highly specific genome editing by both
Cas9 and Cpf1 (Cas12a) nucleases in rice.
Genome Biol 19(1):84. https://doi.org/10.
1186/s13059-018-1458-5
19. Zhang Q, Xing H-L, Wang Z-P et al (2018)
Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis
and its prevention. Plant Mol Biol 96
(4-5):445–456.
https://doi.org/10.1007/
s11103-018-0709-x
20. Doench JG, Hartenian E, Graham DB et al
(2014) Rational design of highly active
sgRNAs for CRISPR-Cas9-mediated gene
inactivation.
Nat
Biotechnol
32
(12):1262–1267. https://doi.org/10.1038/
nbt.3026
21. Wang T, Wei JJ, Sabatini DM et al (2014)
Genetic screens in human cells using the
CRISPR-Cas9
system.
Science
343
(6166):80–84. https://doi.org/10.1126/sci
ence.1246981
22. Liang G, Zhang H, Lou D et al (2016) Selection of highly efficient sgRNAs for CRISPR/
Cas9-based plant genome editing. Sci Rep
6:21451.
https://doi.org/10.1038/
srep21451
23. Liu X, Homma A, Sayadi J et al (2016)
Sequence features associated with the cleavage
efficiency of CRISPR/Cas9 system. Sci Rep
6:19675.
https://doi.org/10.1038/
srep19675
24. Ma X, Zhang Q, Zhu Q et al (2015) A robust
CRISPR/Cas9 system for convenient, highefficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8
(8):1274–1284. https://doi.org/10.1016/j.
molp.2015.04.007
25. Xu H, Xiao T, Chen C-H et al (2015)
Sequence determinants of improved CRISPR
sgRNA
design.
Genome
Res
25
(8):1147–1157. https://doi.org/10.1101/gr.
191452.115
26. Le C, Ran FA, Cox D et al (2013) Multiplex
genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://
doi.org/10.1126/science.1231143
27. Zheng T, Hou Y, Zhang P et al (2017)
Profiling single-guide RNA specificity reveals
a mismatch sensitive core sequence. Sci Rep
7:40638.
https://doi.org/10.1038/
srep40638
28. Nishimasu H, Ran FA, Hsu PD et al (2014)
Crystal structure of Cas9 in complex with
guide RNA and target DNA. Cell 156
(5):935–949. https://doi.org/10.1016/j.cell.
2014.02.001
29. Konermann S, Brigham MD, Trevino AE et al
(2015) Genome-scale transcriptional activation
by an engineered CRISPR-Cas9 complex.
Nature 517(7536):583–588. https://doi.
org/10.1038/nature14136
30. Sander JD, Dahlborg EJ, Goodwin MJ et al
(2011) Selection-free zinc-finger-nuclease
engineering by context-dependent assembly
(CoDA). Nat Methods 8(1):67–69. https://
doi.org/10.1038/nmeth.1542
31. Sanjana NE, Le C, Zhou Y et al (2012) A
transcription activator-like effector toolbox
for genome engineering. Nat Protoc 7
(1):171–192.
https://doi.org/10.1038/
nprot.2011.431
32. Verkuijl SA, Rots MG (2019) The influence of
eukaryotic chromatin state on CRISPR-Cas9
editing efficiencies. Curr Opin Biotechnol
55:68–73.
https://doi.org/10.1016/j.
copbio.2018.07.005
33. Horlbeck MA, Witkowsky LB, Guglielmi B
et al (2016) Nucleosomes impede Cas9 access
to DNA in vivo and in vitro. elife 5:e12677.
https://doi.org/10.7554/eLife.12677
34. Wu X, Scott DA, Kriz AJ et al (2014) Genomewide binding of the CRISPR endonuclease
Cas9 in mammalian cells. Nat Biotechnol 32
(7):670–676. https://doi.org/10.1038/nbt.
2889
35. Daer RM, Cutts JP, Brafman DA et al (2017)
The impact of chromatin dynamics on Cas9mediated genome editing in human cells. ACS
Guidelines for gRNA Design
341