4. Run PCR products on a 0.8% agarose gel (Fig. 9b).
5. For amino acid substitution, the combination of PCR and
restriction enzyme digestion (RFLP) is useful to detect gene
targeting (Fig. 5a, 9c). The procedure is the same as the one
described above (see Subheading 3.1.6, steps 19–23).
6. Both knock-in and substitution GTs are mainly established in
the T1 generation such as line #4, 8, and 12 (Fig. 9b). Then, to
identify heritable knock-in and substitution GTs, an additional
screening and genotyping should be performed in the T2
generation [18].
4 Notes
1. Sometimes actual sgRNA activity might be different from the
efficiency predicted by software. So to avoid the possibility that
mutants will not be obtained due to low Cas9 nuclease activity,
we usually design at least two independent sgRNAs for each
target gene. This enables obtaining different mutant alleles,
and could be a good option to minimize the risk of experiment
failure.
2. The PAM sequence should be not included in the oligomers.
3. If PCR products could not be subcloned into the Zero Blunt
TOPO PCR cloning vector, dilute the concentration of PCR
products 1/5 to 1/20 (or more) with ddH 2 O.
4. In this protocol, AtU6-26 PolIII promoter (At3g13855) was
introduced for multiple targets, a combination of other PolIII
promoters such as AtU3b and At7SL-2 are used to obtain
higher order mutants [10, 11]. The protocol for AtU6-26
described in this chapter is adaptable to other PolIII promoters
(see Subheading 3.1.3).
5. The traditional Cas9 generates DSB at 3 bp upstream of PAM
sequence. Thus short in-del mutations ensue surrounding this
DSB site (Figs. 1b and 8c). On the other hand, ABE and CBE
base editors generate base conversions at positions 3–14 and
4–15 of the protospacer, respectively (Fig. 1b) [20–23]. Thus,
certain restriction enzyme sites must overlap with those possible mutation regions, when the combination of PCR and
restriction enzyme digestion (RFLP) is used for mutant screening. Alternatively, Cas9-induced mutations can be detected by
derived cleaved amplified polymorphic sequences (dCAPS), T7
endonuclease I assay or deep sequencing. The activity window
depends on the base editors and CRISPR/Cas [26]. Thus,
confirmation of base editor activity window should be
priorly done.
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5. For amino acid substitution, the combination of PCR and
restriction enzyme digestion (RFLP) is useful to detect gene
targeting (Fig. 5a, 9c). The procedure is the same as the one
described above (see Subheading 3.1.6, steps 19–23).
6. Both knock-in and substitution GTs are mainly established in
the T1 generation such as line #4, 8, and 12 (Fig. 9b). Then, to
identify heritable knock-in and substitution GTs, an additional
screening and genotyping should be performed in the T2
generation [18].
4 Notes
1. Sometimes actual sgRNA activity might be different from the
efficiency predicted by software. So to avoid the possibility that
mutants will not be obtained due to low Cas9 nuclease activity,
we usually design at least two independent sgRNAs for each
target gene. This enables obtaining different mutant alleles,
and could be a good option to minimize the risk of experiment
failure.
2. The PAM sequence should be not included in the oligomers.
3. If PCR products could not be subcloned into the Zero Blunt
TOPO PCR cloning vector, dilute the concentration of PCR
products 1/5 to 1/20 (or more) with ddH 2 O.
4. In this protocol, AtU6-26 PolIII promoter (At3g13855) was
introduced for multiple targets, a combination of other PolIII
promoters such as AtU3b and At7SL-2 are used to obtain
higher order mutants [10, 11]. The protocol for AtU6-26
described in this chapter is adaptable to other PolIII promoters
(see Subheading 3.1.3).
5. The traditional Cas9 generates DSB at 3 bp upstream of PAM
sequence. Thus short in-del mutations ensue surrounding this
DSB site (Figs. 1b and 8c). On the other hand, ABE and CBE
base editors generate base conversions at positions 3–14 and
4–15 of the protospacer, respectively (Fig. 1b) [20–23]. Thus,
certain restriction enzyme sites must overlap with those possible mutation regions, when the combination of PCR and
restriction enzyme digestion (RFLP) is used for mutant screening. Alternatively, Cas9-induced mutations can be detected by
derived cleaved amplified polymorphic sequences (dCAPS), T7
endonuclease I assay or deep sequencing. The activity window
depends on the base editors and CRISPR/Cas [26]. Thus,
confirmation of base editor activity window should be
priorly done.
144
Daisuke Miki et al.