Design sgRNA pairs using CRoatan

This tool can be used to pick pairs of sgRNAs to target any protein coding human genes. Potent sgRNA pairs are selected based on individual sgRNA predicted efficacy, conservation at target site and prediction of frame shift inducing mutations by micro-homology guided repairs. The search bar below can be used to search for genes, alternatively a table of construct designs for all protein coding human genes can be downloaded here .

Enter/paste gene names (max 1000)

Cloning protocol

To clone pairs of sgRNAs into the pCRoatan dual-sgRNA expressing plasmid, synthetize two oligos of the following sequence:


In both oligos, the Ns are the reverse-complement of the 20bp target sequence. Standard desalted oligos can be used, resuspend the oligos to a final concentration of 100uM in water.

Step 1. Digest 4ug of pCRoatan plasmid with BsmbI for 2 hours at 37C:
4 ug pCRoatan plasmid
5 ul 10x Buffer Tango (Thermo Fischer)
5 ul 10 mM DTT
2 ul BsmBI (Thermo Fischer)
X ul H2O
50 ul Total volume
Step 2. Gel purify the digested plasmid using a QIAquick Gel Extraction Kit. A ~1kb filler should visible on the gel if the digestion was successful. Extract the top, ~7.5kb band.
Step 3. Amplify the dual-promoters using the two oligos as primers:
1 ul 100 uM sgRNA1-oligo
1 ul 100 uM sgRNA2-oligo
1 ul 10 ng pDU6 template
5 ul 10x KOD buffer (Millipore)
5 ul DMSO
4 ul MgSO4
2 ul KOD
X ul H2O
50 ul Total volume

Put the PCR reaction in a thermocycler and run the following program:
  1. 95C for 5 mins
  2. 95C for 30 s
  3. 55C for 30 s
  4. 72C for 30 s
  5. Cycle to step 2 for 20 cycles
  6. 72C for 30 s
Step 4. Purify the PCR reaction using the QIAquick PCR purification kit and digest using BbsI for 2 hours at 37C:
4 ug PCR product
5 ul 10x NEBuffer 2.1 (NEB)
2 ul BbsI (Thermo Fischer)
X ul H2O
50 ul Total volume
Step 5. Gel purify the ~1.2kb PCR product containing the sgRNAs and the two U6 promoters using a QIAquick Gel Extraction Kit.
Step 6. Set up ligation and incubate at room temperature for 1 hour:
X ul 100 ng of digested pCRoatan
X ul 40 ng of digested PCR product
2 ul 10x T4 DNA ligase buffer (NEB)
1 ul T4 DNA ligase (NEB M0202T)
X ul H2O
20 ul Total volume
Step 7. Transform into competent cells. Recombination-deficient bacteria must be used to prevent plasmid recombination. We use Endura electrocompetent cells (Lucigen #60242-2) for this transformation. Plate the transformation on Amp-Zeo-LB low salt agar plates.

Sequence-verified clones

CRoatan Sanger sequence-verified clones can be obtained from TransOMIC technologies, both as one-by-one constructs or as pools. We are currently working on building a complete sequence-verified library with 5 sgRNA pairs for all human genes. The table below summarizes the number of constructs per gene that we have sequenced at this time.
Current number of constructs Number of genes
0 781
1 2318
2 4601
3 5802
4 3140
5 2368