6. We have used 801 bp and 325 bp for 5
0 and 3
0 homology arms
for GFP knock-in into the ROS1 locus, respectively (Fig. 4a),
and the donor construct worked well [18]. However now we
use 800 bp homology arms each for donor.
7. If the products of the second round of PCR are not obtained, a
gradient of annealing temperature PCR (55–70
C) can be
tried. Alternatively, the concentration of first round PCR products can be determined by using the absorption spectrometer,
and an equal amount of PCR products can be used as template
for the second round PCR.
8. As described in Subheading 3.2.4, step 3, the GT-specific
primer set give rise to some false-positive signals that are not
detected by the external primer set (Fig. 9b). However, sometimes it is difficult to amplify both the endogenous and the
knock-in alleles with the external primer sets due to the size of
PCR products and the PCR amplification efficiency. The PCR
conditions for external primer sets should not exceed 25 cycles.
When additional cycles are performed, the band corresponding
to the knock-in allele will not be detected because the PCR
product from the endogenous allele is preferentially amplified.
References
1. Rai A et al (2019) A new era in plant functional
genomics. Curr Opin Syst Biol 15:58–67
2. Alonso JM, Ecker JR (2006) Moving forward
in reverse: genetic technologies to enable
genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536
3. Mba C, Afza R, Bado S, Jian SM (2010)
Induced mutagenesis in plants using physical
and chemical agents. Plant Cell Cult Essent
Methods 20:111–130
4. Gilchrist E, Haughn G (2010) Reverse genetics
techniques: engineering loss and gain of gene
function in plants. Brief Funct Genomics
9:103–111
5. Doudna JA, Charpentier E (2014) Genome
editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1077
6. Cox DB, Platt RJ, Zhang F (2015) Therapeutic
genome editing: prospects and challenges. Nat
Med 21:121–131
7. Chandrasegaran S, Carroll D (2016) Origins of
programmable nucleases for genome engineering. J Mol Biol 428:963–989
8. Weeks DP, Spalding MH, Yang B (2016) Use
of designer nucleases for targeted gene and
genome editing in plants. Plant Biotechnol J
14:483–495
9. Hua K et al (2019) Perspectives on the application of genome editing technologies incrop
breeding. Mol Plant 12:1047–1059
10. Zhang Z et al (2016) A multiplex CRISPR/
Cas9 platform for fast and efficient editing of
multiple genes in Arabidopsis. Plant Cell Rep
35(7):1519–1533
11. Feng Z et al (2018) A highly efficient cell
division-specific CRISPR/Cas9 system generates homozygous mutants for multiple genes in
Arabidopsis. Int J Mol Sci 19:3925
12. Miao C et al (2018) Mutations in a subfamily
of abscisic acid receptor genes promote rice
growth and productivity. Proc Natl Acad Sci
U S A 115:6058–6063
13. Wang ZP et al (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently
generates homozygous mutants for multiple
target genes in Arabidopsis in a single generation. Genome Biol 16:144
14. Mao Y et al (2016) Development of germ-linespecific CRISPR-Cas9 systems to improve the
production of heritable gene modifications in
Arabidopsis. Plant Biotechnol J 14:519–532
15. Eid A, Ali Z, Mahfouz MM (2016) High efficiency of targeted mutagenesis in arabidopsis
via meiotic promoter-driven expression of Cas9
endonuclease. Plant Cell Rep 35:1555–1558
CRISPR/Cas9-Based Genome Editing in Arabidopsis
145
0 and 3
0 homology arms
for GFP knock-in into the ROS1 locus, respectively (Fig. 4a),
and the donor construct worked well [18]. However now we
use 800 bp homology arms each for donor.
7. If the products of the second round of PCR are not obtained, a
gradient of annealing temperature PCR (55–70
C) can be
tried. Alternatively, the concentration of first round PCR products can be determined by using the absorption spectrometer,
and an equal amount of PCR products can be used as template
for the second round PCR.
8. As described in Subheading 3.2.4, step 3, the GT-specific
primer set give rise to some false-positive signals that are not
detected by the external primer set (Fig. 9b). However, sometimes it is difficult to amplify both the endogenous and the
knock-in alleles with the external primer sets due to the size of
PCR products and the PCR amplification efficiency. The PCR
conditions for external primer sets should not exceed 25 cycles.
When additional cycles are performed, the band corresponding
to the knock-in allele will not be detected because the PCR
product from the endogenous allele is preferentially amplified.
References
1. Rai A et al (2019) A new era in plant functional
genomics. Curr Opin Syst Biol 15:58–67
2. Alonso JM, Ecker JR (2006) Moving forward
in reverse: genetic technologies to enable
genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536
3. Mba C, Afza R, Bado S, Jian SM (2010)
Induced mutagenesis in plants using physical
and chemical agents. Plant Cell Cult Essent
Methods 20:111–130
4. Gilchrist E, Haughn G (2010) Reverse genetics
techniques: engineering loss and gain of gene
function in plants. Brief Funct Genomics
9:103–111
5. Doudna JA, Charpentier E (2014) Genome
editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1077
6. Cox DB, Platt RJ, Zhang F (2015) Therapeutic
genome editing: prospects and challenges. Nat
Med 21:121–131
7. Chandrasegaran S, Carroll D (2016) Origins of
programmable nucleases for genome engineering. J Mol Biol 428:963–989
8. Weeks DP, Spalding MH, Yang B (2016) Use
of designer nucleases for targeted gene and
genome editing in plants. Plant Biotechnol J
14:483–495
9. Hua K et al (2019) Perspectives on the application of genome editing technologies incrop
breeding. Mol Plant 12:1047–1059
10. Zhang Z et al (2016) A multiplex CRISPR/
Cas9 platform for fast and efficient editing of
multiple genes in Arabidopsis. Plant Cell Rep
35(7):1519–1533
11. Feng Z et al (2018) A highly efficient cell
division-specific CRISPR/Cas9 system generates homozygous mutants for multiple genes in
Arabidopsis. Int J Mol Sci 19:3925
12. Miao C et al (2018) Mutations in a subfamily
of abscisic acid receptor genes promote rice
growth and productivity. Proc Natl Acad Sci
U S A 115:6058–6063
13. Wang ZP et al (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently
generates homozygous mutants for multiple
target genes in Arabidopsis in a single generation. Genome Biol 16:144
14. Mao Y et al (2016) Development of germ-linespecific CRISPR-Cas9 systems to improve the
production of heritable gene modifications in
Arabidopsis. Plant Biotechnol J 14:519–532
15. Eid A, Ali Z, Mahfouz MM (2016) High efficiency of targeted mutagenesis in arabidopsis
via meiotic promoter-driven expression of Cas9
endonuclease. Plant Cell Rep 35:1555–1558
CRISPR/Cas9-Based Genome Editing in Arabidopsis
145