Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.


Construction of the cosmid containing four and eight gRNA units
For construction of the cosmid containing four gRNA units, 2 g of SwaI-linearized pAxc4wit2 was ligated at 15 C overnight with 300 ng of head-block in a volume typically of 15 L. For eight gRNA units, 300 ng each of head-and mid-blocks were used for ligation. Very high concentration of these fragments, a minimum volume, and also high purity were important. Half of the ligated DNA was digested with SwaI in a volume of 70 L to remove the colonies containing the parent pAxc4wit2 cosmids lacking the insert and only 1 L was used for packaging. The lambda packaging kits used were Lambda Inn (Nippon Gene, Japan) or Gigapack Plus (Stratagene). A scale of one-fifth using the former kit was usually sufficient, thereby reducing the cost, which is comparable with transformation. The orientation of the array of eight gRNA units was examined by digestion of the cosmid with EcoRV. The entire sequences were determined using 12 primers listed in Supplementary Table S1.
To prepare a 50-mL scale culture, 1 L of the miniprep cosmid DNA was directly used for lambda packaging: though miniprep cosmid DNA is circular, twentieth of the packaging DNA usually yielded about 10 3 colonies. Culture flasks were vigorously shaken to achieve good aeration as usually applied for preparation. Cosmid DNA was prepared using an alkali-lysis procedure. 1 In the step of phenol-chloroform extraction the DNA solution was shaken vigorously to remove thoroughly contaminated DNase, which in our experience is more important than taking care of mechanical breaking of the cosmids. The drying-up step of plasmid preparation was avoided. Therefore, after centrifugation of the ethanol precipitation, the remaining 70% ethanol on the cosmid pellet was removed with a piece of Kimwipe, and TE was immediately added. Handling of the cosmids was essentially the same as that of the plasmids, except that described above. Throughout the cosmid experiments whole colony was suspended in miniculture for miniprep to minimize the duplication times of the E. coli DH5 . In addition, scissor-cut tips of micropipettes except miniprep DNAs for checking of their structures were used throughout the handling of cosmid DNAs to avoid mechanical breaking of cosmid DNA, especially for concentrated or ligated cosmids. Cosmids were always stored at 20 C.
A very high concentration was critical for lambda packaging because tetramolecular ligation of the insert flanked with two cos-containing vectors is needed, while ligation for typical plasmid cloning involves a dimolecular reaction between the insert and the vector plasmid DNA. Tetramolecular ligation for construction of cosmid containing eight gRNA units can be performed efficiently because the packaging efficiency is extremely high unless a very high concentration is achieved. SwaI recognizes 8 nt of ATTT/AAAT and produces blunt ends. Practically, any DNA fragment, unless it contains a SwaI site, can be cloned to the SwaI site, after the treatment of Klenow enzyme, ligation and the subsequent recleavage with SwaI to remove the clones lacking the insert. Therefore, the SwaI site might be more convenient than the polylinker consisting of multiple enzyme sites. All the terminal sequences of the head and mid-blocks are PvuII/HincII/BsaHI because of the usage of primers *ampl Head-A F, *ampl Head-E R, *ampl Mid-A F, and *ampl Mid-E R for construction. PvuII and HincII produce a blunt end and can be cloned to SwaI site at the E4 cloning position of pAxc4wit2, while BsaHI produces 5 -CG overhang, which can be ligated with ClaI site at the E1 cloning position of pAxc4wit2. AlwNI connects Head and Mid blocks. The gRNA/U6 junctions of cassettes B, C, and D are polylinker sequences that are not homologous to either U6 promoter or gRNA scaffold. Therefore, they can be used as unique sequencing primers (seq-B F/R, seq-C F/R, and seq-D F/R) and also allow easy subcloning of desired single or double units. When eight gRNA units are included in the cosmid, it cannot directly be sequenced because primers in the cassette B, C and D are present twice. Thus, before sequencing, the head and mid-block were separately amplified using the forward and reverse primers containing AlwNI and AdV primers outside the array of eight gRNA units.
The Unit positions, the name of oligos and their sequences are shown. In these oligonucleotides spaces are introduced according to Figure 2B, though the direction is from 5' to 3' in the reverse strand. U1 means unit 1, etc.; HBc, HBV genotype C; t and b, top and bottom strands; F and R, forward and reverse orientation based on that of U6 promoter.
pParent-Head-A (=pbs-Head-A), cassette fragment head-A (BglI-BsaI 521bp)    Figure S2. A convenient construction of cosmid containing four gRNA units. (a) The strategy for the construction of the cosmids. Head t1+t2 fragment (third row) was constructed by five fragment ligation (first row) and amplification using the amplifying forward primer of head-A cassette and the reverse sequencing primer of cassette C containing an NsiI site (second row, *ampl Head-A F and seq-C R-primers, respectively). Similarly, the head t3+t4 fragment (sixth row) was constructed using the forward sequencing primer of cassette C containing the NsiI site and the reverse primer of head-E cassette (fifth row, seq-C F and *ampl Head-E R-primers, respectively). Sequences of the primers are shown in Supplementary Table S1. (b)(c) Ligation patterns of the three cassettes and two targets. Production of fragments containing two gRNAs is easier than that constructing a four gRNA-containing fragment as described in Fig. 1c and 1d. The desired ligation products containing cassette fragments of head-A, B, and C with targets 1 and 2 and cassette fragments of C, D, and head-E with targets 3 and 4 were abundant and easily visible in the ligation step. (b, left) Ligation pattern of head-A, B, C, t1, and t2. In this figure, t1 and t2 are ignored; for example, B means sole B, t1-B, B-t2, and t1-B-t2. The desired fragment of 1.3 kb was observed as a very dense band. The bands above 1.3 kb were generated because head-A fragment was obtained by the digestion of BsaI and EcoRV, which produces blunt ends. Therefore, head-A can be ligated with another head-A in the reverse orientation, forming CBA-ABC, for example. Off-target t1-2 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGtcgtaaagtctcttaggtgtcagg-3'