CRISPR/Cas-Mediated Base Editing: Technical Considerations and Practical Applications

Base editing represents a new dimension of CRISPR/Cas-mediated precise editing to generate single-nucleotide changes in DNA or RNA independently of double-strand breaks and homology-directed repair. Since its invention in 2016, many base-editing tools have been developed to install point mutations in a diverse array of animal, plant, and microbial organisms. Base editing yields a high efficiency of editing with very low rates of indel formation. Rapid advances in base-editing techniques have significantly reduced unintended editing and expanded the scope and utility of genome targeting. Base Editors work in both dividing and non-dividing cells and can be applied to correct 61% of human pathogenic mutations listed in the ClinVar database. Base editing has drawn great academic and industrial interest because it is broadly applicable to basic research, synthetic biology, therapeutics, and crop improvement. Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture. Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture. a range of bases in the protospacer sequence which is favorable for the editing activity of a BE. Activity windows vary across base-editing platforms. Most BEs have an activity window of about 5–6 nt. composed of catalytically impaired nuclease and laboratory evolved DNA-adenosine deaminase, ABEs convert a targeted A–T base pair to a G–C base pair by deaminating adenosine in the DNA. a CRISPR/Cas-mediated genome-editing method that uses a combination of a catalytically impaired nuclease and a nucleotide deaminase to introduce a point mutation at a target locus in DNA or RNA. clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9), a two-component genome editing system including a single-guide RNA (sgRNA) and a Cas9 nuclease. A predesigned sgRNA directs Cas9 to bind to and cut a DNA sequence upstream of the PAM. composed of a catalytically impaired nuclease and cytosine deaminase, CBEs convert a targeted C–G base pair to a T–A base pair by deaminating cytosine in DNA. catalytically dead Cas9, developed by mutating both the nuclease (RuvC and HNH) domains of Cas9. dCas9 lacks DNA-cleavage activity but retains RNA-directed DNA-binding activity. a molecular method that makes specific changes in a genome by the deletion, insertion, or replacement of a fragment or specific bases of the genome, which allows the precise removal, addition, or alteration of genetic material. insertion (in) or deletion (del) of nucleotides in genomic DNA. Genomic cleavage by Cas9 or other nucleases is followed by indel generation. Indels generally cause frameshift mutations except when the length of the indel is 3 nt or multiple of 3 nt. nickase-Cas9, a mutated form of a Cas9 that creates a nick in the target DNA instead of a DSB. Mutation in either the RuvC or HNH domains of Cas9 generates a nCas9. BEs generally use nCas9-D10A (mutated RuvC). any unintended editing that occurs due to nonspecific interaction of nucleases at sites other than the targeted site in DNA or RNA. Recognition of non-canonical PAM and partial homology of the guide RNA sequence with nontarget sequence may lead to off-target editing. a short sequence (2–6 bp) of nucleotides situated immediately adjacent to the target DNA sequence and which is essential for target recognition by CRISPR-associated nucleases. For most discovered nucleases, PAM is present downstream from the target sequence, although some (e.g., Cas12a) can have an upstream PAM. developed by combining catalytically inactive nuclease (dCas13b) and ‘adenosine deaminase acting on RNA’, RBEs convert a targeted A base to an I base in RNA.