Raviraj Banakar1,2,3, Mollie Schubert4, Michael Collingwood4, Christopher Vakulskas4, Alan L Eggenberger1,2, Kan Wang5,6. 1. Department of Agronomy, Iowa State University, Ames, IA, USA. 2. Crop Bioengineering Center, Iowa State University, Ames, IA, USA. 3. Present Address: Department of Plant and Microbial Genomics, University of Minnesota, St Paul, MN, 55108, USA. 4. Integrated DNA Technologies, Coralville, IA, USA. 5. Department of Agronomy, Iowa State University, Ames, IA, USA. kanwang@iastate.edu. 6. Crop Bioengineering Center, Iowa State University, Ames, IA, USA. kanwang@iastate.edu.
Abstract
BACKGROUND: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site. FINDINGS: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. CONCLUSIONS: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.
BACKGROUND: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the ricephytoene desaturase (PDS) gene as a target site. FINDINGS: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. CONCLUSIONS: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.
Authors: Raviraj Banakar; Mollie Schubert; Gavin Kurgan; Krishan Mohan Rai; Sarah F Beaudoin; Michael A Collingwood; Christopher A Vakulskas; Kan Wang; Feng Zhang Journal: Front Genome Ed Date: 2022-01-12
Authors: Wannaporn Ittiprasert; Chawalit Chatupheeraphat; Victoria H Mann; Wenhui Li; André Miller; Taiwo Ogunbayo; Kenny Tran; Yousef N Alrefaei; Margaret Mentink-Kane; Paul J Brindley Journal: Int J Mol Sci Date: 2022-01-06 Impact factor: 5.923