CRISPR-Cas9 DNA Base-Editing and Prime-Editing
Abstract
:1. Introduction
2. CRISPR Genome Editing
3. DNA Base-Editing
3.1. Cytosine Base-Editors
3.2. Adenine Base-Editors
3.3. Prime-Editing
4. Potential Applications of DNA Base-Editors and Prime-Editors
4.1. Editing Large Genes
4.2. Targeting Autosomal Dominant Diseases
4.3. Editing Premature Stop Codons
4.4. Editing Splice-Site Variants
4.5. Current Limitations
5. Delivery Systems
Adeno-Associated Vectors
6. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AAG | Alkyl adenine DNA glycosylase |
AAP | Assembly activating protein |
AAV | Adeno-associated virus |
ABE1.2 | First-generation adenine deaminase |
ABE7.10 | Seventh-generation adenine base-editor |
ABE8 | Eighth-generation adenine base-editor |
AID | Activation-induced cytidine deaminase |
APOBEC1 | Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1 |
AsCpf1 | Acidaminococcus sp. Cfp1 |
LbCpf1 | Lachnospiraceae bacteriumare Cpf1 |
Bps | Base-pairs |
SaCas9 | Staphylococcus aureus Cas9 |
BE | Base-editor |
BER | Base excision repair |
Cas | CRISPR-associated genes |
CBE1 | First-generation cytosine base-editor |
CBE2 | Second-generation cytosine base-editor |
CBE4 | Fourth-generation cytosine base-editor |
CjCas9 | Campylobacter jejuni Cas9 |
Cpf1/Cas12a | Prevotella and Francisella1 |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
dAsCpf1 | Catalytically inactive AsCpf1 |
dCas9 | Catalytically deficient Cas9 |
dLbCpf1 | Catalytically inactive LbCpf1 |
DSB | Double-strand DNA break |
CBE | Cytosine base-editor |
ABE | Adenine base-editor |
PE | Prime editor |
Indel | Insertion or deletion |
NHEJ | Non-homologous end joining |
GDS | Gene delivery system |
HDR | Homology directed repair |
HEK293 | Human embryonic kidney cells |
HF | High-fidelity version of dCas9 |
HT1 | Hereditary tyrosinemia type 1 |
iPSCs | Induced pluripotent stem cells |
ITR | Inverted terminal repeats |
M-MLV RT | Moloney murine leukemia virus reverse transcriptase |
nCas9 | Cas9 nickase |
NLS | Nuclear Localization Signal |
ORF | Open reading frame |
PACE | Phase assisted continuous evolution |
PAM | Protospacer adjacent motif |
gRNA | Guide RNA |
PANCE | Phage-assisted non-continuous evolution |
PBS | Primer binding site |
PE1 | First-generation prime editor |
pegRNA | Prime editing guide RNA |
pmCDA1 | Petromyzon marinus cytidine deaminase 1 |
rAAV | Recombinant AAV |
RK | Rhodopsin kinase |
RP | Retinitis pigmentosa |
RT | Reverse Transcriptase |
ScCas9 | Streptococcus canis Cas9 |
sgRNA | Small guide RNA |
SpCas9 | S. pyogenes’ Cas9 |
Spy-mac | Cas9 fusion derived from Streptococcus pyogenes and Streptococcus macacae |
TadA | tRNA adenosine deaminase |
TALEN | Transcription activator-like effector nucleases |
tracrRNA | Trans-activating RNA |
UNG | Uracil N-glycosylate |
ZFN | Zinc-finger nuclease |
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Base-Editor | Architecture | Editing Efficiency 1 | Notes | Refs |
---|---|---|---|---|
BE1 | rAPOBEC1-dCas9 | 0.8–7.7% in human cells | First-generation BE | [46] |
BE2 | rAPOBEC1-dCas9-UGI | Up to 20% | Prefers TC motifs | [46] |
HF2-BE2 | rAPOBEC1-HF2 nCas9-UGI | 11.6–50% | Prefers TC motifs | [47,48] |
BE3 | rAPOBEC1-SpnCas9-UGI | Varies widely by cell type & target genes | Prefers TC motifs | [46,47,49,50,51,52,53,54,55,56,57,58,59,60,61,62] |
HF-BE3 | rAPOBEC1-HFnCas9-UGI | 21 ± 3% | Reduced off-target editing | [52] |
YE1-BE3 | rAPOBEC1 (W90Y, R126E) SpnCas9-UGI | Comparable to BE3 | Narrowed editing window | [63] |
EE-BE3 | rAPOBEC1 (R126E, R132E) SpnCas9-UGI | Comparable to BE3 | Narrowed editing window | [63] |
YEE-BE3 | rAPOBEC1 (W90Y, R126E, R132E)-SpnCas9-UGI | Comparable to BE3 | Narrowed editing window | [63] |
VQR-BE3 | rAPOBEC1-VQR SpnCas9-UGI | 14.5–52% | Expanded PAM targeting | [63] |
EQR-BE3 | rAPOBEC1-EQR SpnCas9-UGI | 7.5–35% | Expanded PAM targeting | [63] |
VRER-BE3 | rAPOBEC1-VRER SpnCas9-UGI | 11–32% | Expanded PAM targeting | [63] |
SaKKHBE3 | rAPOBEC1-KKH SanCas9-UGI | 14–62% | Expanded PAM targeting | [63] |
FNLS-BE3 | rAPOBEC1-SpnCas9-UGI | 41–93% | Additional N-terminus NLS; Increased editing efficiency | [62] |
RA-BE3 | rAPOBEC1 (RA)-SpnCas9-UGI | 30–58% | Increased editing efficiency | [62] |
A3A-BE3 | hAPOBEC3A-SpnCas9-UGI | 22.5% | Preferential deamination of cytidines in a TCR motif | [59] |
eA3A-HF1-BE3-2xUGI | APOBEC3A-HF1 SpnCas9-UGI-UGI | 17.5% | Deaminates cytosines with preference TCR > TCY > VCN; Increased editing efficiency | [59] |
eA3A-Hypa-BE3-2xUGI | APOBEC3A-Hypa SpnCas9-UGI-UGI | 14% | Deaminates cytosines with preference TCR > TCY > VCN; Increased editing efficiency | [59] |
hA3A-BE3 | hAPOBEC3A-SpnCas9-UGI | 2–62% | Efficient editing in methylated region and in GpC context | [64] |
hA3B-BE3 | hAPOBEC3B-SpnCas9-UGI | 2–62% | Intermediate editing efficiency | [64] |
hA3G-BE3 | hAPOBEC3G-SpnCas9-UGI | 2–62% | Greatly decreased editing efficiency | [64] |
hAID-BE3 | hAPOBEC3A-SpnCas9-UGI | 2–62% | Intermediate editing efficiency | [64] |
SaCas9-BE3 | rAPOBEC1-SanCas9-UGI | ∼50–75% | Expanded targeting range | [63] |
xCas9-BE3 | rAPOBEC1-xnCas9-UGI | 37% (NGG PAM) | Expanded targeting range | [60] |
ScCas9-BE3 | rAPOBEC1-ScnCas9-UGI | 19–41% | Affinity to minimal 5′-NNG-3′ PAM sequences | [65] |
SniperCas9-BE3 | rAPOBEC1-SnipernCas9-UGI | 0.04–50% | Increased sgRNA scope; further reduced off-target activities | [33] |
iSpyMac-BE3 | rAPOBEC1-iSpyMacnCas9-UGI | 50% | Elevated editing efficiencies on 5′-NAAN-3′ targets | [66] |
Target-AID | SpnCas9-CDA1-UGI | 17–55% | First-generation base-editor | [8] |
Target-AID-NG | SpnCas9 (NG)-CDA1-UGI | 1–38% | Expanded targeting range | [67,68] |
CRISPR-X | SpdCas9-MS2-hAID | N/A | High activity; used for random mutagenesis | [69] |
TAM | SpdCas9-hAID (P182X) | N/A | High activity; used for random mutagenesis | [70] |
BE-PLUS | SunTag-SpnCas9-scFv-rAPOBEC1-UGI | 2–38% | Broadened targeting window; reduced off-target editing | [71] |
BE4 | rAPOBEC1-SpnCas9-UGI-UGI | Varies widely by cell type & target genes | Increased editing efficiency | [47,49,61,72] |
BE4-Gam | Gam-rAPOBEC1-SpnCas9-UGI-UGI | 17–58% | Increased editing efficiency and product purity | [49,61] |
BE4-Max | rAPOBEC1-SpnCas9-UGI-UGI | 69–77% | Codon optimized for mammalian cells | [72] |
AncBE4-Max | rAPOBEC1-SpnCas9-UGI-UGI | 75–84% | Ancestral reconstruction of the deaminase component | [72] |
SaCas9-BE4 | rAPOBEC1-SanCas9-UGI-UGI | 25–60% | Expanded PAM targeting | [49] |
SaCas9-BE4-Gam | Gam-rAPOBEC1-SanCas9-UGI-UGI | 42–67% | Increased editing efficiency and product purity | [49] |
evoBE4max | rAPOBEC1-SpnCas9-UGI-UGI | Up to plateau levels (~60–80%) | Improved efficiency in GC context | [73] |
evoFERNY-BE4max | rAPOBEC1-SpnCas9-UGI-UGI | Up to plateau levels (~60–80%) | 29% smaller than APOBEC1 | [73] |
Cas12a-BE | rAPOBEC1-dLbCpf1-UGI | 3–46% | Can target T-rich PAM sequence | [74] |
ABE7.8/9/10 | ecTadA-ecTadA *-SpnCas9 | 1.7–20% | First generation ABE | [13] |
xCas9-ABE7.10 | ecTadA-ecTadA *-nxCas9 | 69% (NGG PAM) | Expanded PAM targeting | [60] |
VQR-ABE | ecTadA-ecTadA *-Sp VQR nCas9 | 20% | Expanded PAM targeting | [75] |
Sa(KKH)-ABE | ecTadA-ecTadA *-Sa KKH nCas9 | 16% | Expanded PAM targeting | [75] |
ABEmax | ecTadA-ecTadA *-SpnCas9 | 27–52% | Improved editing efficiency | [72] |
ABE7.10max | ecTadA-ecTadA *-SpnCas9 | 19.2–40.7% | Improved editing efficiency | [76] |
ABE8e | ecTadA-ecTadA *-SpnCas9 | 18%–86% | Improved editing efficiency | [77] |
PE1 | dSpCas9-MMLV-RT | 0.7–5.5% | First generation PE | [10] |
PE2 | dSpCas9-MMLV-RT | 1.6- to 5.1-fold improvement over PE1 | Targets all transition/transversion mutations; small indels | [10] |
PE3 | nSpCas9-MMLV-RT | 20–50% | Targets all transition/transversion mutations; small indels | [10] |
CRISPR-Tool | Function | Gene Size (kb) |
---|---|---|
SpCas9 | Nuclease | 4.2 |
SaCas9 | Nuclease | 3.2 |
CjCas9 | Nuclease | 2.9 |
xCas9 | Nuclease | 3.7 |
AsCpf1 | Nuclease | 3.9 |
LbCpf1 | Nuclease | 3.7 |
rAPOBEC1 | Cytosine Deaminase | 0.7 |
ecTadA(8e)-dimer | Adenine Deaminase | 1.2 |
MMLV RT | Reverse Transcriptase | 2.2 |
UGI | Inhibits UNG | 0.3 |
BE4 | Cytosine Base-editor | 5.6 |
ABE7.10 | Adenine Base-editor | 5.3 |
PE2 | Prime Editor | 6.4 |
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Kantor, A.; McClements, M.E.; MacLaren, R.E. CRISPR-Cas9 DNA Base-Editing and Prime-Editing. Int. J. Mol. Sci. 2020, 21, 6240. https://doi.org/10.3390/ijms21176240
Kantor A, McClements ME, MacLaren RE. CRISPR-Cas9 DNA Base-Editing and Prime-Editing. International Journal of Molecular Sciences. 2020; 21(17):6240. https://doi.org/10.3390/ijms21176240
Chicago/Turabian StyleKantor, Ariel, Michelle E. McClements, and Robert E. MacLaren. 2020. "CRISPR-Cas9 DNA Base-Editing and Prime-Editing" International Journal of Molecular Sciences 21, no. 17: 6240. https://doi.org/10.3390/ijms21176240