Prykhozhij, S. V. | Search bioRxiv

bioRxiv2021

CRISPR Knock-in Designer: automatic oligonucleotide design software to introduce point mutations using CRISPR/Cas9

Knock-in of precise point mutations into protein-coding genes has been one of the earliest and most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. The ability to perform such precise gene editing is crucial to interrogate the function of specific protein residues and to create models of human diseases caused by protein amino acid changes. The homologous protein residues can be mutated in model animal species, and the consequences of these mutations can be studied, leading to a better understanding of the disease in question. Design of point mutation knock-in strategies has been a combination of manual steps assisted by several computational tools resulting in a time-consuming process and preventing a single rapid and integrated solution. We have therefore designed CRISPR Knock-in Designer, which can perform rapid and automatic design of point mutation knock-in DNA oligonucleotides upon provision of the mutation, a guide RNA, and essential identifier or sequence information. The tool supports most experimentally established CRISPR types and has multiple options for the resulting oligonucleotides to satisfy the needs of most users. We also provide allele-specific PCR-based and restriction enzyme-based genotyping strategies as part of the program output. CRISPR Knock-in Designer adjusts to the genomic context of any target codon and tries to design knock-in strategies when a codon straddles two exons, a situation we explored in whole genomes of several model species. CRISPR Knock-in Designer output can also be adapted for use with some of the newer Prime Editing design tools to facilitate the introduction of a specific mutation sequence using this advanced technology.

genetics↗

bioRxiv2017

Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

Single-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

molecular biology↗