getLibraryOligoCas12a6plexgRNA: Function to assemble oligo for pooled synthesis for Cas12a...

In chris-hsiung/bears01: What the Package Does (One Line, Title Case)

Function to assemble oligo for pooled synthesis for Cas12a 6-plex gRNA expression. The oligo will consist of: Adaptor5p-BsmbI5p-pos1spacer-DR1-pos2spacer-DR16-pos3spacer-DR18-pos4spacer-DR10-pos5spacer-DR3-pos6spacer-BsmbI3p-Adaptor3p. DR sequences are taken from Deweirdt et al., Nat. Biotech 2020. Assumes the oligo will be PCR'd using user-provided adaptor sequences, then digested with BsmbI and ligated into backbone. The BsmbI5p and BsmbI3p sites are hard-coded to be compatible with vector backbone containing 5' DR and 3' DR sequences, which are not encoded in the oligo itself (e.g. compatible with vectors designs of pRG212, pCH39, pCH49, etc.).

Description

Function to assemble oligo for pooled synthesis for Cas12a 6-plex gRNA expression. The oligo will consist of: Adaptor5p-BsmbI5p-pos1spacer-DR1-pos2spacer-DR16-pos3spacer-DR18-pos4spacer-DR10-pos5spacer-DR3-pos6spacer-BsmbI3p-Adaptor3p. DR sequences are taken from Deweirdt et al., Nat. Biotech 2020. Assumes the oligo will be PCR'd using user-provided adaptor sequences, then digested with BsmbI and ligated into backbone. The BsmbI5p and BsmbI3p sites are hard-coded to be compatible with vector backbone containing 5' DR and 3' DR sequences, which are not encoded in the oligo itself (e.g. compatible with vectors designs of pRG212, pCH39, pCH49, etc.).

Usage

getLibraryOligoCas12a6plexgRNA(
  pos1name = "holder",
  pos1spacer,
  pos2name = "holder",
  pos2spacer,
  pos3name = "holder",
  pos3spacer,
  pos4name = "holder",
  pos4spacer,
  pos5name = "holder",
  pos5spacer,
  pos6name = "holder",
  pos6spacer,
  Adaptor5p,
  Adaptor3p,
  sep = "_",
  outputdir = getwd()
)

Arguments

pos1name, pos2name, pos3name, pos4name, pos5name, pos6name, sep	optional string containing name for each spacer that will be separated by 'sep' if returning a name for the oligo is desired. Names defaults to 'holder' if not supplied.
pos1spacer, pos2spacer, pos3spacer, pos4spacer, pos5spacer, pos6spacer	string containing only spacer DNA sequences of length 19-23nt. Must not contain BsmbI sites. Must not contain TTTT. Must not start with TTT, because the last base of each DR is a T and that would form an undesirable TTTT at the junction.
Adaptor5p, Adaptor3p	string containing PCR adaptor sequences to be used for subpool amplification (recommend taking from https://weissman.wi.mit.edu/crispr/) The final oligo is checked for undesirable BsmbI sites (in excess of the 2 that should be there); if fails, throws error.

Value

A list containing oligoname and oligosequence. oligoname is in the format 'pos1name_pos2name_pos3name_pos4name_pos5name_pos6name'.

Author(s)

Chris Hsiung

chris-hsiung/bears01 documentation built on April 9, 2024, 2:01 a.m.