or one 1 nt RNA bulge within the “core” region is sufficient to
prevent cleavage. If at least three mismatches in total are present, additional bulges should abolish cleavage.
9. Efficient gRNAs show only few interactions between the guide
sequence and gRNA backbone. Furthermore, intact stem-loop
structures are crucial for high activity.
5 Conclusion
Many considerations and useful tools are available to aid for the
selection of suitable CRISPR targets. However, a considerable
inconsistency is reported between experimental systems and currently available prediction tools are far from predicting gRNA
efficiency with high fidelity. While transient protoplast assays can
also give relatively high levels of confidence, experimental validation inducing heritable changes in individuals remains the only way
to achieve certainty regarding gRNA efficiency.
References
1. Urnov FD (2018) Genome editing
B.C. (Before CRISPR): lasting lessons from
the “old testament”. CRISPR J 1(1):34–46.
https://doi.org/10.1089/crispr.2018.29007.
fyu
2. Jinek M, Chylinski K, Fonfara I et al (2012) A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/
10.1126/science.1225829
3. Doudna JA, Charpentier E (2014) Genome
editing the new frontier of genome engineering with CRISPR-Cas9. Science 346
(6213):1258096. https://doi.org/10.1126/
science.1258096
4. Knott GJ, Doudna JA (2018) CRISPR-Cas
guides the future of genetic engineering. Science 361(6405):866–869. https://doi.org/
10.1126/science.aat5011
5. Schindele P, Wolter F, Puchta H (2018) Transforming plant biology and breeding with
CRISPR/Cas9, Cas12 and Cas13. FEBS Lett
592(12):1954–1967.
https://doi.org/10.
1002/1873-3468.13073
6. Kumlehn J, Pietralla J, Hensel G et al (2018)
The CRISPR/Cas revolution continues: from
efficient gene editing for crop breeding to plant
synthetic biology. J Integr Plant Biol 60
(12):1127–1153. https://doi.org/10.1111/
jipb.12734
7. Cebrian-Serrano A, Davies B (2017) CRISPRCas orthologues and variants: optimizing the
repertoire, specificity and delivery of genome
engineering tools. Mamm Genome 28
(7–8):247–261. https://doi.org/10.1007/
s00335-017-9697-4
8. Fu Y, Foden JA, Khayter C et al (2013) Highfrequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells. Nat
Biotechnol 31(9):822–826. https://doi.org/
10.1038/nbt.2623
9. Hsu PD, Scott DA, Weinstein JA et al (2013)
DNA targeting specificity of RNA-guided Cas9
nucleases. Nat Biotechnol 31(9):827–832.
https://doi.org/10.1038/nbt.2647
10. Fu Y, Sander JD, Reyon D et al (2014)
Improving CRISPR-Cas nuclease specificity
using truncated guide RNAs. Nat Biotechnol
32(3):279–284. https://doi.org/10.1038/
nbt.2808
11. Lin Y, Cradick TJ, Brown MT et al (2014)
CRISPR/Cas9 systems have off-target activity
with insertions or deletions between target
DNA and guide RNA sequences. Nucleic
Acids Res 42(11):7473–7485. https://doi.
org/10.1093/nar/gku402
12. Ren X, Yang Z, Xu J et al (2014) Enhanced
specificity and efficiency of the CRISPR/Cas9
system with optimized sgRNA parameters in
Drosophila. Cell Rep 9(3):1151–1162.
https://doi.org/10.1016/j.celrep.2014.09.
044
13. Zhang J-P, Li X-L, Neises A et al (2016) Different effects of sgRNA length on CRISPR340
Patrick Schindele et al.
prevent cleavage. If at least three mismatches in total are present, additional bulges should abolish cleavage.
9. Efficient gRNAs show only few interactions between the guide
sequence and gRNA backbone. Furthermore, intact stem-loop
structures are crucial for high activity.
5 Conclusion
Many considerations and useful tools are available to aid for the
selection of suitable CRISPR targets. However, a considerable
inconsistency is reported between experimental systems and currently available prediction tools are far from predicting gRNA
efficiency with high fidelity. While transient protoplast assays can
also give relatively high levels of confidence, experimental validation inducing heritable changes in individuals remains the only way
to achieve certainty regarding gRNA efficiency.
References
1. Urnov FD (2018) Genome editing
B.C. (Before CRISPR): lasting lessons from
the “old testament”. CRISPR J 1(1):34–46.
https://doi.org/10.1089/crispr.2018.29007.
fyu
2. Jinek M, Chylinski K, Fonfara I et al (2012) A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/
10.1126/science.1225829
3. Doudna JA, Charpentier E (2014) Genome
editing the new frontier of genome engineering with CRISPR-Cas9. Science 346
(6213):1258096. https://doi.org/10.1126/
science.1258096
4. Knott GJ, Doudna JA (2018) CRISPR-Cas
guides the future of genetic engineering. Science 361(6405):866–869. https://doi.org/
10.1126/science.aat5011
5. Schindele P, Wolter F, Puchta H (2018) Transforming plant biology and breeding with
CRISPR/Cas9, Cas12 and Cas13. FEBS Lett
592(12):1954–1967.
https://doi.org/10.
1002/1873-3468.13073
6. Kumlehn J, Pietralla J, Hensel G et al (2018)
The CRISPR/Cas revolution continues: from
efficient gene editing for crop breeding to plant
synthetic biology. J Integr Plant Biol 60
(12):1127–1153. https://doi.org/10.1111/
jipb.12734
7. Cebrian-Serrano A, Davies B (2017) CRISPRCas orthologues and variants: optimizing the
repertoire, specificity and delivery of genome
engineering tools. Mamm Genome 28
(7–8):247–261. https://doi.org/10.1007/
s00335-017-9697-4
8. Fu Y, Foden JA, Khayter C et al (2013) Highfrequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells. Nat
Biotechnol 31(9):822–826. https://doi.org/
10.1038/nbt.2623
9. Hsu PD, Scott DA, Weinstein JA et al (2013)
DNA targeting specificity of RNA-guided Cas9
nucleases. Nat Biotechnol 31(9):827–832.
https://doi.org/10.1038/nbt.2647
10. Fu Y, Sander JD, Reyon D et al (2014)
Improving CRISPR-Cas nuclease specificity
using truncated guide RNAs. Nat Biotechnol
32(3):279–284. https://doi.org/10.1038/
nbt.2808
11. Lin Y, Cradick TJ, Brown MT et al (2014)
CRISPR/Cas9 systems have off-target activity
with insertions or deletions between target
DNA and guide RNA sequences. Nucleic
Acids Res 42(11):7473–7485. https://doi.
org/10.1093/nar/gku402
12. Ren X, Yang Z, Xu J et al (2014) Enhanced
specificity and efficiency of the CRISPR/Cas9
system with optimized sgRNA parameters in
Drosophila. Cell Rep 9(3):1151–1162.
https://doi.org/10.1016/j.celrep.2014.09.
044
13. Zhang J-P, Li X-L, Neises A et al (2016) Different effects of sgRNA length on CRISPR340
Patrick Schindele et al.