Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

Abstract

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration

“…Several studies have attempted to find more available PAM sequences, but they could not completely eliminate the sequence restrictions. Kleinstiver developed variants of the CRISPR enzyme to eliminate the PAM requirement, but not using the PAM can reduce the accuracy and cause off-targeting ( 30 , 31 ). In addition to studies targeting the Cas nuclease itself, several nucleic acid technologies have played a role.…”

Integrative dissection of gene regulatory elements at base resolution

“…Critical bases and functional motifs will be missed as a consequence. Base editors with less restrictive PAM site requirements (Rosello et al, 2022) could improve the resolution of future screens. While base editor approaches are mainly focused on C-to-T or A-to-G transitions, prime editors could enable more systematic base changes if they could be applied at scale (Anzalone et al, 2019).…”

A minimally invasive fin scratching protocol for fast genotyping and early selection of zebrafish embryos

“…Circa 1 nl of a mix containing 40 pmol/μL of sgRNA (crRNA + tracrRNA) and the 500 ng/μl mRNA encoding ancBE4max were injected into 1 cell stage embryos. Fish were screened for occurrence of pigmentation defects upon 48-hour post fertilization (hpf) following Rosello et al, 2022 24 . Stable plekhh1 knocked out sh line was previously generated in Del Bene laboratory via standard CRISPR/Cas9 technique by injection of two sgRNAs (formed starting from the crRNA oligos 5'-GCATATGAAACTCCCGGTCC-3' and 5'-ATCGTGCCACAGTCTCGTCC-3', Alt-R® CRISPR-Cas9 crRNA, IDT, Integrated DNA Technologies) together with Cas9 protein at 15 µM (IDT).…”

“…To this aim, we performed FS-based genotyping on F0 obtained using CRISPR-Cas9 DNA base-editing technology 50 that produces C:G to T:A conversions into the genome 51,52 recently implemented in zebra sh by Del Bene's team to introduces a premature STOP codon in the tyrosinase (tyr, W273*). The resulting reduced function of the tyr enzyme generated partially depigmented mutants in F0, which were easily selected under the microscope (Figure 2d,e) and served as positive crispant controls whose mutation could also be detected via Sanger sequencing 23,24 (Figure 2d-g).…”

“…Transient or stable transgenic sh labeling speci c cell populations and tissues or subcellular structures are readily and e ciently produced by transposon-mediated transgenesis and TALENs-or CRISPR/Cas-based gene knock-out or knock-in are nowadays routinely used to generate disease models [19][20][21] . In addition, gene editing techniques for targeted modi cations are being established in this model [22][23][24] and promise to generate precise and possibly personalized disease models. Of note, an extensive collection of lines is available to researchers at certi ed international or European stock centers 6,25 .…”