83cells or directly in mouse embryos to ensure sufficient activity of the
sgRNA. The principles for the donor design are: (1) to include the intended
mutation, as well as silent mutations based on similar codon usages, to block
sgRNA re-targeting; (2) to introduce new restriction enzyme sites via silent
mutations for easy genotyping, particularly useful for identification of point
or small mutations. Among more than 70 projects we have performed, we
failed only one due to the low complexity of the homologous arms. The
single- strand donor oligo can be designed in several different ways (e.g. PAM
vs. non-PAM strand, symmetric vs. asymmetric homologous arms, etc.), and
we prefer to follow an asymmetric design on a non-PAM strand [ 44 , 45 ].- For larger gene knock-ins using a donor plasmid, a sufficient sgRNA cutting
activity is very critical for successful targeting. Therefore, validation of the
sgRNA’s activity prior to the construction of the donor plasmid is necessary.
The donor plasmid is then designed based on the sgRNA selection, because
the insertion site should ideally be placed near the sgRNA cut site. The length
of the homologous arms is also an important factor. We recommend having
a total length of at least 4 kb (e.g. 2 + 2 kb or 1.5 + 2.5 kb) of homologous
arms. An even longer length is required when the homologous arms contain a
significant portion of repeats, particularly the transposable elements and tan-
dem repeats. The plasmid should be injected in the circular form because it
significantly reduces the toxicity and the rate of random integration compared
to the linearized form. - For generation of the conditional allele, we use a strategy similar to that of
the large knock-in project. We design a pair of non-overlapping adjacent
sgRNAs to target each desired loxP insertion site, so that a total of four
sgRNAs are used for an efficient deletion between two loxP insertion sites in
the genome. The donor plasmid is constructed to contain the floxed exon and
flanking homologous arms, but the sequence encompassing the sgRNA rec-
ognition sites is disrupted by the loxP sequence to avoid the sgRNA re-
targeting. The conditional allele can also be made by inserting the loxP
sequence to the DNA simultaneously or sequentially using two sgRNAs and
corresponding single-strand donor oligos. The frequency of the former is
quite low because it requires two knock-in events to happen in the same
embryos and in cis. The outcome is unpredictable and usually takes a lot more
rounds of injections and resources to achieve it. We recommend doing
the latter by inserting the first loxP and then breeding the one-loxP mice for
targeting the second loxP, which allows the mice to be made in a more
predictable way.
- Use of optimized sgRNA scaffold. sgRNA contains a ~20 nucleotide user-defined
target sequence, followed by the scaffold that consists of a duplex and three
stem-loop structures. The sgRNA scaffold is essential for Cas9 binding and the
full catalytic activity of the complex [ 7 , 8 , 46 , 47 ]. Because the optimized scaf-
fold was reported to further enhance the sgRNA activity [ 48 , 49 ], we modified
our sgRNA scaffold accordingly by flipping an A-U base pair and extending the4 A Transgenic Core Facility’s Experience in Genome Editing Revolution