Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing

Abstract

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

“…2023. 65 In detail: Three plasmids for expression of MAD7 were designed by Jedrzejczyk et al. 2022: 1×NLS (pNic28-EcMad7-1×NLS), 3×NLS (pNic28-EcMad7-3×NLS) and 4×NLS (pNic28-EcMad7-4×NLS).…”

Cas12a and MAD7, genome editing tools for breeding

“…CRISPR-Cas12a nuclease MAD7, which was released by Inscripta Inc., is an engineered Cas12a variant originating from Eubacterium rectale found on the island of Madagascar. It shares 76% identical nucleotides with the native form (Liu et al 2020b, Rojek et al 2023, Vanegas et al 2023. It encodes a monomeric 147.9 kDa polypeptide consisting of 1,263 amino acids.…”

“…It encodes a monomeric 147.9 kDa polypeptide consisting of 1,263 amino acids. Notably, MAD7 shares just 31% amino acid homology with the canonical Acidaminococcus Cas12a (also known as AsCpf1) (Lin et al 2021, Liu et al 2020b, Price et al 2020, Rojek et al 2023.…”

“…MAD7 shows a preference for 5ʹ-YTTN-3ʹ PAM sites, making it available for genome editing in T-rich DNA sequences, whereas PAM sites of Cas9 are in the form 5ʹ-NGG-3ʹ, making it useful in G-rich DNA sequences (Mund et al 2023, Price et al 2020, Rojek et al 2023. Mad7 crRNAs are designed with 5ʹ direct repeats of either 21 or 35 nucleotides, followed by a 21-nucleotide protospacer region at the 3ʹ end.…”

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

“…Although not yet extensively used in CHO cell line engineering, the reported improved efficiency and lower cytotoxicity of Cas9 nickase and dCas9 PmCDA1-mediated base editing compared to Cas9 KO [34] may lead to the broader exploitation of this approach in CHO cell line engineering in the future. Moreover, the CRISPR toolbox in CHO cell line engineering has recently been expanded to include the engineered Cas variants Mad7, also known as ErCas12a, in CHO cells [166].…”