Generation of genetically modified mice using SpCas9-NG engineered nuclease

Abstract Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.

Download Full-text

Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

Scientific Reports ◽

10.1038/s41598-021-89546-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuu Asano ◽

Kensuke Yamashita ◽

Aoi Hasegawa ◽

Takanori Ogasawara ◽

Hoshie Iriki ◽

...

Keyword(s):

Genome Editing ◽

Dictyostelium Discoideum ◽

Streptococcus Pyogenes ◽

Nucleotide Substitution ◽

Point Mutations ◽

Targeted Mutagenesis ◽

Single Amino Acid ◽

Specific Gene ◽

Knock Out ◽

Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Download Full-text

Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

Scientific Reports ◽

10.1038/s41598-020-74960-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Samy Alghadban ◽

Amine Bouchareb ◽

Robert Hinch ◽

Polinka Hernandez-Pliego ◽

Daniel Biggs ◽

...

Keyword(s):

Mouse Models ◽

High Efficiency ◽

Genetically Modified ◽

Delivery Methods ◽

Embryo Survival ◽

Guide Rnas ◽

Standard Tool ◽

Survival And Development ◽

Effective Delivery ◽

Mouse Zygotes

Abstract CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.

Download Full-text

Generation of Genetically Modified Mice Using the CRISPR–Cas9 Genome-Editing System

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot090704 ◽

2016 ◽

Vol 2016 (2) ◽

pp. pdb.prot090704 ◽

Cited By ~ 26

Author(s):

Jorge Henao-Mejia ◽

Adam Williams ◽

Anthony Rongvaux ◽

Judith Stein ◽

Cynthia Hughes ◽

...

Keyword(s):

Genome Editing ◽

Genetically Modified ◽

Genetically Modified Mice

Download Full-text

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

Journal of Visualized Experiments ◽

10.3791/60808 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hirofumi Nishizono ◽

Mohamed Darwish ◽

Hideki Uosaki ◽

Nanami Masuyama ◽

Motoaki Seki ◽

...

Keyword(s):

High Efficiency ◽

Genetically Modified ◽

Genetically Modified Mice

Download Full-text

Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.618385 ◽

2020 ◽

Vol 2 ◽

Author(s):

Chengwei Zhang ◽

Guiting Kang ◽

Xinxiang Liu ◽

Si Zhao ◽

Shuang Yuan ◽

...

Keyword(s):

Genome Editing ◽

Plant Species ◽

Streptococcus Pyogenes ◽

Genome Engineering ◽

Human Cells ◽

Rice Plants ◽

Potential Target ◽

Protospacer Adjacent Motif ◽

Target Sites ◽

Related Plant

The CRISPR-Cas9 system enables simple, rapid, and effective genome editing in many species. Nevertheless, the requirement of an NGG protospacer adjacent motif (PAM) for the widely used canonical Streptococcus pyogenes Cas9 (SpCas9) limits the potential target sites. The xCas9, an engineered SpCas9 variant, was developed to broaden the PAM compatibility to NG, GAA, and GAT PAMs in human cells. However, no knockout rice plants were generated for GAA PAM sites, and only one edited target with a GAT PAM was reported. In this study, we used tRNA and enhanced sgRNA (esgRNA) to develop an efficient CRISPR-xCas9 genome editing system able to mutate genes at NG, GAA, GAT, and even GAG PAM sites in rice. We also developed the corresponding xCas9-based cytosine base editor (CBE) that can edit the NG and GA PAM sites. These new editing tools will be useful for future rice research or breeding, and may also be applicable for other related plant species.

Download Full-text

Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽

10.3390/cells10082099 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2099

Author(s):

Yunxing Liu ◽

Fang Liang ◽

Zijiong Dong ◽

Song Li ◽

Jianmin Ye ◽

...

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Gene Editing ◽

Zebrafish Genome ◽

Human Cells ◽

Ribonucleoprotein Complex ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

Download Full-text

Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

10.1101/620351 ◽

2019 ◽

Author(s):

Pranam Chatterjee ◽

Noah Jakimo ◽

Joseph M. Jacobson

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Sequence Space ◽

Target Site ◽

Gene Repression ◽

Single Base ◽

Base Editing ◽

Homology Directed Repair ◽

Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

Download Full-text

Orthogonal CRISPR-Cas genome editing and efficient inhibition with anti-CRISPRs in zebrafish embryos

10.1101/2020.11.07.372151 ◽

2020 ◽

Author(s):

Paige R. Takasugi ◽

Evan P. Drage ◽

Sahar N. Kanishka ◽

Marissa A. Higbee ◽

James A. Gagnon

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

High Efficiency ◽

Bacterial Species ◽

Zebrafish Embryos ◽

Type Ii ◽

Guide Rnas ◽

Highly Effective ◽

Orthogonal Systems ◽

Spatiotemporal Control

AbstractThe CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system limits options for multiplexed editing. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpCas9), Streptococcus aureus (SaCas9), and Lachnospiraceae bacterium (LbCas12a, previously known as LbCpf1) are highly effective, orthogonal systems capable of operating simultaneously in zebrafish. We also demonstrate that type II Acrs are effective inhibitors of SpCas9 in zebrafish. These results indicate that at least three orthogonal CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing in zebrafish.

Download Full-text

Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Science ◽

10.1126/science.aba8853 ◽

2020 ◽

Vol 368 (6488) ◽

pp. 290-296 ◽

Cited By ~ 95

Author(s):

Russell T. Walton ◽

Kathleen A. Christie ◽

Madelynn N. Whittaker ◽

Benjamin P. Kleinstiver

Keyword(s):

High Resolution ◽

Genome Editing ◽

Streptococcus Pyogenes ◽

Genetic Variants ◽

Human Cells ◽

Target Site ◽

Protospacer Adjacent Motif ◽

Wide Range ◽

Almost All ◽

Site Recognition

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Download Full-text