RESUMO
Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA de Cadeia Simples/genética , Animais , Éxons/genética , Feminino , Edição de Genes/métodos , Técnicas de Introdução de Genes , Genes Reporter/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Zigoto/fisiologiaRESUMO
An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR typeâ V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the inâ vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirusâ 16 (HPV-16) and parvovirusâ B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.
Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas CRISPR-Cas/genética , DNA Viral/química , Papillomavirus Humano 16/genética , Ácidos Nucleicos Imobilizados/química , Parvovirus/genética , Acidaminococcus/genética , Técnicas Biossensoriais , Clivagem do DNA , Técnicas Eletroquímicas , Eletrodos , Humanos , Limite de Detecção , Células-Tronco Mesenquimais , Sensibilidade e Especificidade , Propriedades de Superfície , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismoRESUMO
CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements. Here, we harness six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that remarkably increase the targeting range in the genomes. Moreover, we identify optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a.