Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

Schubert MS, Thommandru B, Woodley J, Turk R, Yan S, Kurgan G, McNeill MS, Rettig GR.
Source: Scientific Reports
Publication Date: (2021)
Issue: 11(1): 19482
Cells used in publication:
Species: human
Tissue Origin: kidney
Species: human
Tissue Origin: cervix
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

Delivery of genome editing reagents by nucleofection. Electroporation was performed using the Lonza™ Nucleofector™ 96-well Shuttle™ System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1× phosphate bufered saline (PBS) and resuspended in 20 µL of solution SF or SE (Lonza). Cell suspensions were combined with RNP complex(es), Alt-R Cas9 or Cpf1 (Cas12a) Electroporation Enhancer (Integrated DNA Technologies) and HDR donor template (if applicable). Tis mixture was transferred into one well of a Nucleocuvette™ Plate (Lonza) and electroporated using manufacturer’s recommended protocols (except for HEK293, which used protocol 96-DS-150). Afer nucleofection, 75 µL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 µL was transferred to a 96-well culture plate with 175 µL pre-warmed culture media.


CRISPR-Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines.