ViroBytes

Introduction

ViroBytes is a modified BioBytes method for "shuffling" of AAV Cap genes. Main motivation for the new protocol is the insufficient incorporation of certain serotypes (especially AAV 5) observed in standard shuffling techniques (Grimm et al., 2008). The principle of BioByte formation and annealing persists. Sticky overhangs are used for selective combination of the bytes but a different method is used for the production of individual ViroBytes.

Five AAV serotypes (1,2,5,6,8 and 9) were selected due to their different infection and transduction properties and the Cap genes sequences were compared. The analysis revealed multiple homology regions which were used for rational fragment formation. Total number of fragments per Cap gene is eight in our case and all fragments have similar length around ~250bp to assure similar behaviour in the ligation reaction. The Bytes are created and amplified by PCR using Cap- and FragmentX-specific primers. These primers contain flanking recognition sequence for Bsa1 precisely positioned in front of and behind the homology regions so that the enzyme cuts in the homology regions and forms unique sticky ends with 4nt overhangs. This procedure allows us to avoid arduous design of overhangs with incorporated uracils further used for USER^TM digestion. Also for the purposes of shuffling, the homology regions need to be exploited in order to avoid frame shifts which cannot be easily accomplished using standard BioByte protocol.

Important facts

BsaI recognition and cutting sequence:

  5'...GGTCTCN/NNNN...3'
  3'...CCAGAGNNNNN/...5'

hence overhang: 5'-NNNN

Homology region used:

between Anchor and Fragment1:

  5'...ATGG...3'
  3'...TACC...5'

between Fragment1 and Fragment2 (bp 279-303 relative to AAV1 Cap):

  5'...GGAG...3'
  3'...CCTC...5'

between Fragment2 and Fragment3 (bp 688-716):

  5'...GACA...3'
  3'...CTGT...5'

between Fragment3 and Fragment4 (bp 1013-1016):

  5'...AACC...3'
  3'...TTGG...5'

between Fragment4 and Fragment5 (bp 1282-1285):

  5'...CAGC...3'
  3'...GTCG...5'

between Fragment5 and Fragment6 (bp 1444-1447):

  5'...AACT...3'
  3'...TTGA...5'

between Fragment6 and Fragment7 (bp 1740-1743):

  5'...GTGG...3'
  3'...CACC...5'

between Fragment7 and Fragment8 (bp 2040-2043):

  5'...TCAC...3'
  3'...AGTG...5'

ViroByte design

• Anchor:

5'-Biotin-tctctctctctctctaagctt gtctgagtgactagcattcg ttaattaac+5nt(cap specific)

- first 15nt are to form ssDNA linker (improves efficiency of Anchor binding onto Steptavidin-coated magnetic beads)

- italicized is HindIII recognition sequence

- bold is amplification sequence

- bold italicized is PacI recognition sequence

• Anchor complement:

5'-gttaattaa cgaatgctagtcactcagac aagctt

- reverse complimentary strand to Anchors

• Fragment X forward primers:

5'-gctacggtctcaNNNN+~20-25nt Cap specific

- bold is BsaI recognition sequence

- NNNN = cap specific homology sequence from all cap genes (also cutting sequence of BsaI)

• Fragment X reverse primers:

5'-gctacggtctcgNNNN

Protocols

Anchor preparation

References

Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, Kay MA. In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses. J Virol 2008;82:5887–5911.