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1、1、经典的基因编辑原理Genomic DMAilsgRNA (tracrRNA-crRNA chimerai【i-nTn mNon-homologous end joining (NHEJ)I山 lIL 川 LI1LIHII口111】1帀|-|11门丁1!工Homology directed repair tHDBSite-specific DNA breakFWM sequenceDonor DMAThe CRISPR-associated endonuclease Cas9 could target specific DNA loci and make double-strand brea
2、ks (DSBs) under the guidance of the sgRNA. The presence of the Protospacer Adjacent Motif (PAM) at the target sequence is mandatory for successful Cas9 binding and catalysis a protection mechanism to avoid self-cutting in CRISPR- containing organisms. DNA double strand breaks can be repaired by homo
3、logous recombination (HR) or non-homologous end joining (NHEJ). NHEJ re-ligates DSBs in an error-prone manner allowing for insertion or deletion of several bases at the DSB site. HR, on the hand,is a high-fidelity repair mechanism that uses an identical (or very similar) copy of the DSB region as a
4、template for repair.操作方法：两步法转染，先转染Cas9蛋白，形成表达Cas9蛋白的细胞系，再 转入靶向特定基因的sgRNA即可。特点是效率较高。一步法转染：转入同时表达Cas9与sgRNA的质粒。使其靶向特定的基因即可。特点是耗时较短上述两种方法都利用了细胞本身的非同源重组修复。其效率较高，缺点是无法进行精确修复。而转入带有同源臂的片段，即可实现同源重组修复。特点是精确基因修复，缺点是效率较低。操作更复杂。2、升级版的基因编辑(单基因碱基编辑)Unlike mutagens causing random nucleobase deamination, RNA-progra
5、mmable deaminase systems, composed of a catalytically impaired CRISPRCas9 variant (D10A Cas9 nickase (nCas9) or catalytically deficient D10A/H840A Cas9 (dCas9) from S. pyogenes) and a deaminase protein from various sources, enable single-nucleotide conversions or base editing in cells and organisms
6、in a gRNA-dependent manner. These base editing systems can be divided into two categories: cytosine base editors (CBEs) that convert a C:G base pair to a T:A pair and ABEs that convert a A:T pair to a G:C pair.2.1胞嘧啶碱基编辑(靶向链进行编辑，CtT或者GtA)5KTEN LinkerCBE1（胞嘧啶碱基编辑）第一代CBEls rAPQBECl-XTEN-dCas9需要dCas9与s
7、gRNA加上XTEN连接一个胞嘧啶脱氨酶。即可对基因进行定点dCas9tytidrne DeaminaseGuide RNA(gKNA)编辑De 口mination window4-S23Target (20 bp)PAMAAAGUGGCACCGA -I I I I I I IC /SXUUUUUUCGUGGCIL*5Z JGTCACCTCAATGACTAGGGGUUUUAGAGCUAGaJI/IH* 1111 AelJUCAACUAUJGCCUGALJCGGAAiy(AAAUU CGAUA III* GAA3AGGACAKGAlTqACCTCpAATGACTAGGGTGG5CAACCAC l
8、llllllirkllI I I l I I I I I ITCCTGTAGCTACAGTGGAGGTTACTGATCCACCCpTGGTG(typically, positions 12 to 17 (suboptimal) or positions 14 to 17 (optimal) upstream of a protospacer adjacent motif (PAM) sequence in the single-stranded, non-target DNA strand exposed for the deaminase reaction at a target proto
9、spacer site编辑位置在 PAM前 12-17 碱基处发生。体外编辑效率在 50-80%之间大致流程图如下5rDNA扩3F5tiTTTh乡CytidineDeamination of target CdCasEiGenomic DNABase-edited DNAGukieRNA(bRNA)Genomicbinding a：4and opening亍DNAreplication or repair但是存在一个问题就是基因的本身的修复将导致其效率低下。而其主要作用的是细胞中的一个糖苷酶作用。(Cellular DNA repair response to U:G heteroduplex
10、 DNA was responsible for the large decrease in base editing efficiency in cells。)CBE2（胞嘧啶碱基编辑）第二代Uracil DNA glycosylase inhibitor (UGI)：An 83-residue protein from Bacillus subtilisbacteriophage PBS1，Potently blocks human UDG activity 。加上一个 UGI 糖苷酶抑制基因。使其效率提升了 3 倍左右CBE3（胞嘧啶碱基编辑）第三代3Cas9 NickasedCa$9(
11、A84CH)CytidineDeaminaseKTEN LinkerGuide RNA fj JgKNA将 dCas9 换成能发生剪切的 dCas9 ，只能对非互补配对链进行剪切，使其形成一 个缺口，便于 DNA 修复。这个系统的好处在于，第一：可以对 sgRNA 靶序列前面的 5 个碱基进行编辑。从（第48bp）处。第二，还可以将dCas9换成其余的蛋白，其实识别位点PAM可 以多样化。这样的好处在于能编辑的范围更大。Cas9 Nickase ModuleseditorCa$9 ModulePAMCBE3Streptococci pyogenes Cas9 (5p匚NGGVQR-CBE3SV
12、QR,-as9NGANEQR-CBE 菇EQR-CasSNGAGVRER-CBE3SVRER-Cas9NGCGSaCBESSStaphylococcusCa$9 (SCasS)NNGRRTSaKKH-CBE3SSaCas9 variant(three mutations)NNNRRTCBE4（胞嘧啶碱基编辑）第四代匚BE4： Garn rAPOBECl-XTEN-Ca59 Nickase-UGS-UGI在CBE3的基础上再加一个UGI以及噬菌体Mu蛋白Gam,其目的在于保护 双链断裂后不被降解，促进连接修复。使其基因编辑效率在CBE3基础上提升了 50%。Gam: Binding to the
13、 ends of DSBs and protects them from degradation. Base editors nick the strand opposite the U, cleavage of the glycosidic bond by UNG followed by processing of the resulting AP site by AP lyase would result in a DSB, which promotes indel formation2.2腺嘌呤碱基编辑（非靶向链进行编辑，AtG或者TtC）其原理与 CBE 系统大同小异需要一个可以发生单
14、链剪切的dCas9,加上脱氧腺苷脱氨酶（TadA）,核定位信匸戲g Nidca&e Module Bind arxl open gancmic DNA Pratogpaoer PAM 宀3阴 ANAIgRNAIDaminat&Genomic DNAtarget A in ssDNA bubbleNick non-dited slrnd眾:皿jnummi 11111111111111r号，加上sgRNA即可针对基因进行单碱基突变。（脱氨酶与Cas9剪切酶（Cas9n） 的N末端融合，在gRNA弓导下，非靶向链中的腺苷脱氨，暴露为单链DNA， 然后将腺嘌吟A转化为肌苷I,肌苷在DNA水平被当作G进
15、行读码复制，然而细胞内对于肌苷的切除修复并不敏感，因此，ABEs能够高效地产生A/T到G/C的突变，且能维持较高的产物纯度。）如下图所示bct5-3I DNA nepair or + FEplicaljon llllllllllilllllTniTlirJ?. u i ii , i, iii I n i亠juBase-cditsd DINA特点是非靶向链进行AG或者TC的替换。位置在靶位点起始处4-7bp处， 编辑的宽度在4-6bp左右。编辑效率都大于99%。(the activity windows of are approximately 4-6 nucleotides wide, from protospacer positions 4-7 for ABE7.10 ， Very High Product Purity (Typically 99.9%)， Very Low Rates Of Indels (Typically 0.1%)
