{'fraction_non_alphanumeric': 0.02696871628910464, 'fraction_numerical': 0.003236245954692557, 'mean_word_length': 5.008639308855291, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Advances and applications of CRISPR/Cas toolbox

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) is an adaptive immune system in bacteria or archaea that responds to the invasion of foreign DNA or RNA. It detects and destroys target invasive nucleic acid through crRNA and Cas protein. The CRISPR/Cas system has been modified to a variety of gene-editing tools, and due to its accessibility and ease of use, it has rapidly become the most popular approach for genome engineering. The discovery of different classes of Cas proteins has broadened the applications of CRISPR/Cas for gene-editing. For example, Cas9 and Cas12 can be applied to recognize and edit target DNA sequence, while Cas13 and RCas9 can be applied to recognize and edit target RNA sequence. To make the CRISPR/Cas system more useful for gene editing, many efforts have been undertaken to increase its on-target editing efficiency, decrease the off-target effects, and reduce the size of Cas proteins for easy delivery to tissues and cells. Furthermore, nuclease-deficient Cas9 (dCas) proteins have been fused to different types of effector proteins to perform more functions. For example, for precise gene editing, the base editor (dCas fused to deaminase) can be used for single-base DNA editing, cytosine base editors achieving C to T (G to A) substitutions and adenine base editors achieving A to G (T to C) substitutions; and the prime editor (dCas fused to reverse transcriptase) can be used for single-base DNA editing or small DNA insertion and deletion. For regulating gene expression, CRISPRa and CRISPRi (dCas fused to a transcriptional activation or repression motif) can be used to activate or inhibit gene expression. In addition, CRISPR/Cas has also been applied for functional genetic screening, target DNA or RNA detection, DNA or RNA live imaging, and cell lineage tracing. The CRISPR/Cas-based tools improve our understanding of biological mechanisms and have also been applied for gene therapy in some clinical trials, such as editing genes or gene regulatory elements for curing thalassemia, sickle cell disease and Leber congenital amaurosis 10, and transforming antigens or deleting negative regulators genes for T-cell immunotherapy. In this review, we discuss the different types of CRISPR/Cas systems, the development and improvement of CRISPR/Cas based gene editing tools and the applications of these tools in scientific research. We also discuss the applications of CRISPR/Cas in clinical therapy and use β-hemoglobin diseases as an example to show the advantages and accessibility of CRISPR/Cas based gene therapy. Taking above, the CRISPR/Cas toolbox could be a key to the scientific research and the next generation of gene therapies.