The complete CRISPR guide: from mechanism to NGS analysis

You've heard that CRISPR can edit any gene in any organism. That's basically true. But there's a big gap between knowing that CRISPR exists and actually designing experiments, validating edits, and analyzing your sequencing data with confidence.

This series closes that gap.

CRISPR from Bench to Analysis is a 22-post series that takes you from the molecular mechanism of Cas9 all the way through running your own NGS analysis pipeline. No assumed background in bioinformatics. Just clear explanations, real tools, and runnable code where it matters.

How this series works

The series is divided into three arcs. Each arc ends at a milestone — a point where you've built a real, usable skill and could stop if that's all you needed.

Posts go live every week. Bookmark this page and check back, or follow the blog so you don't miss one.

Arc 1 — Plan your experiment

You'll finish this arc knowing: How to choose the right CRISPR editing system for your goal, design validated guide RNAs (including pegRNAs for prime editing and base editing guides), and predict off-target sites in silico before you ever touch a cell.

This arc covers the decision-making that happens before your experiment starts — the part that saves you from spending three weeks on a guide that was never going to work.

#	Post	Status
1	How CRISPR-Cas9 actually works: mechanism, PAM sites, and DNA repair	✅ Published
2	Cas12a (Cpf1) vs Cas9: which one should you use?	✅ Published
3	Base editing explained: making precise changes without cutting DNA	✅ Published
4	Prime editing: how it works and when to use it over Cas9	✅ Published
5	What makes a good gRNA: Cas9 and Cas12a guide design principles	Coming Mar 19
6	Designing gRNAs for base editing: hitting the right editing window	Coming Mar 26
7	Designing pegRNAs for prime editing: tools and principles	Coming Apr 2
8	In silico off-target prediction: how the algorithms work and which tools to trust	Coming Apr 9
9	Choosing your CRISPR system and designing validated guides: an end-to-end walkthrough	Coming Apr 16

Arc 2 — Run and measure

You'll finish this arc knowing: How to assess editing efficiency and specificity using the right method for your experiment — and what each result actually means.

This arc is about the validation step that too many people skip or get wrong. T7E1, TIDE, amplicon NGS, GUIDE-seq — you'll know what each one measures, when to use it, and what it can't tell you.

#	Post	Status
10	On-target assessment methods compared: T7E1, TIDE, and amplicon NGS	Coming Apr 23
11	T7E1 and Surveyor assays: how they work, their limits, and how to interpret results	Coming Apr 30
12	Sanger + TIDE/ICE: quantifying CRISPR editing without NGS	Coming May 7
13	Amplicon NGS for CRISPR: how to design your sequencing strategy	Coming May 14
14	Detecting off-targets in the lab: GUIDE-seq, CIRCLE-seq, and Digenome-seq compared	Coming May 21
15	Measuring CRISPR editing efficiency from start to finish: a complete protocol overview	Coming May 28

Arc 3 — Analyze your data

You'll finish this arc knowing: How to go from raw FASTQ files to a complete editing report — including on-target efficiency, indel spectra, and off-target quantification. You'll have run real pipelines with real tools.

This is the arc that turns a wet-lab CRISPR experiment into a complete, publishable dataset.

#	Post	Status
16	Understanding CRISPR amplicon sequencing reads: what the data actually tells you	Coming Jun 4
17	CRISPResso2: the standard tool for CRISPR NGS analysis (full tutorial)	Coming Jun 11
18	CRISPECTOR: quantifying on- and off-target editing from paired NGS samples	Coming Jun 18
19	MAGeCK: analyzing pooled CRISPR screens from raw counts to gene hits	Coming Jun 25
20	CRISPR NGS tools compared: CRISPResso2 vs CRISPECTOR vs MAGeCK vs Debase	Coming Jul 2
21	Building a complete CRISPR analysis pipeline: from FASTQ to editing report	Coming Jul 9
22	The full CRISPR experiment analyzed: from guide design to NGS interpretation	Coming Jul 16

Where should you start?

Never heard of CRISPR? Start at Post 1. It covers the Cas9 mechanism from scratch — no prior knowledge needed.

Know the basics but want to design better experiments? Posts 5–8 (Arc 1) cover guide design, off-target prediction, and tool comparisons. Jump straight there.

Already running CRISPR experiments and want to validate them better? Arc 2 (Post 10) is your entry point — it starts with a comparison of all the assessment methods so you can figure out which one fits your situation.

Have sequencing data and don't know what to do with it? Go straight to Arc 3 (Post 16).

A few things to know about this series

All software tools covered are free to use. Every bioinformatics package in this series — CRISPResso2, MAGeCK, CRISPECTOR — is open-source. The design tools (CRISPOR, CHOPCHOP, PrimeDesign) are free web tools. You don't need a paid subscription for anything covered here.

This series covers SpCas9 as the baseline. Cas12a, base editors, and prime editors are all covered in Arc 1. When a principle only applies to one system, it's clearly labeled.

Code is always runnable. Every command and script in Arc 3 is tested and complete. Copy, paste, run — it should work, and when something might break, I'll tell you why.

Want to go deeper? The book CRISPR from Bench to Analysis goes beyond the blog — full wet lab protocols, decision flowcharts, complete pipelines, and troubleshooting guides. Get it on Ko-fi →

Starting from scratch? Post 1 covers the Cas9 mechanism — it's the foundation everything else builds on.

What are you most hoping to learn from this series? Drop a comment below — it helps me know where to focus the most detail.