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To examine the length of HDR template homology sequences required for Cas9-mediated HDR, we tested four single-stranded and two double-stranded HDR templates for EMX1 bearing homology arms ranging from 30 to 250 nt in length (template 2 7 in Figure 3A).
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All those conforming, at least in part, to the selected templates (1kil, 1s35, 1sum, 1txd, 2boq, 2i0m, 2of3) were used in further analysis, along with five models produced by PROTINFO [40] from the two full length templates (1sum, 2i0m).
Templates sequenced 40 72 times averaged 109 bp in length; templates sequenced 20 40 times averaged 148 bp; and templates sequenced 5 20 times were an average of 286 bp (data not shown).
The resulting collection of 14,227 sequences represented my full-length template database.
On average the resulting alignments were between 0.61% worse and 0.34% better than the alignments generated by SILVA when using full-length template sequences.
Thus, the replicated products were predominantly the same length as the full-length template.
Only 22 transmembrane beta-strands were detected when the full-length template sequence was analyzed.
To determine the specificity of the reactions, all reaction conditions were tested against a purified full-length template as well as other non-specific splice variants.
A DBN is constructed using a fixed-length template which is unrolled in order to model a sequence of any arbitrary length.
When searching against full-length templates, 7 to 9-mers provided the closest matches.
To assemble the full-length templates, the PCR products encoding Leishmania Rabs were fused by PCR with the universal fragment 5 (Fig. S2A), while the fragments containing mammalian Rab were fused with fragment 6 (Fig. S2D).
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